Phosphine derivatives of fluorescent compounds

ABSTRACT

A phosphine derivative of DyLight dyes modified with ethylene glycol or (poly)ethylene glycol groups. In one embodiment, the compounds are useful in chemoselective ligation reactions.

This application is a continuation of U.S. patent application Ser. No.13/546,483 filed Jul. 11, 2012, which claims priority to U.S.Provisional Application No. 61/507,742 filed Jul. 14, 2011, each ofwhich is expressly incorporated by reference herein in its entirety.

A phosphine derivative of DyLight dyes modified with ethylene glycol or(poly)ethylene glycol groups. Phosphine derivatives of DyLight 550, 650,and 755, which contain ethylene glycol or (poly)ethylene glycol, aredisclosed. The phosphine derivatives are useful in chemoselectiveligation reactions, also termed bioorthogonal reactions, e.g., withazido groups to form a covalent bond, with applications to “Click”chemistry producing reactions that are high yielding, wide in scope,creating only byproducts that can be removed without chromatography, arestereospecific, are simple to perform, and can be conducted in easilyremovable or benign solvents, as known to one skilled in the art.

Compounds useful as labels with properties comparable to knownfluorescent compounds are disclosed. The compounds can be conjugated toproteins and nucleic acids for biological imaging and analysis.Synthesis of the compounds, formation and use of the conjugatedcompounds, and specific non-limiting examples of each are disclosed.

Compounds that react with biomolecules (e.g., antigens, antibodies,DNA-segments with the corresponding complimentary species for measuringenzyme kinetics, receptor-ligand interactions, nucleic acidhybridization kinetics in vitro as well as in vivo, etc.), termed labelsor dyes, are useful for, e.g., pharmacological characterization ofreceptors and drugs, binding data, etc. Compounds such as xanthyliumsalts (U.S. Pat. No. 5,846,737) and/or cyanines (U.S. Pat. No.5,627,027) are used for such applications, but aggregate and formdimers, especially in aqueous solution, due to planarity of theirπ-system. Compounds that have insufficient hydrophilicity undergonon-specific interactions with various surfaces, resulting in problemswhen attempting purify the corresponding conjugate, and anunsatisfactory signal to noise ratio.

Efforts are directed to reducing undesirable properties by introducingsubstituents that increase the hydrophilicity of the compounds. Forexample, sulfonic acid function substituents have been introduced intothe cyanine chromophore. U.S. Pat. No. 6,083,485 (Licha) and U.S.application Ser. Nos. 09/968,401 and 09/989,853 (Molecular Probes)disclose cyanine compounds having one of the common methyl groups in the3-position of the terminal indole heterocycle substituted by anω-carboxyalkyl function, and in which the previously present (e.g. inCy3 or Cy5) N-alkyl or N-ω-carboxyalkyl functions are replaced byN-ω-alkyl sulfonic acid functions. WO 05/044923 discloses cyaninecompounds having the common methyl substituent in the 3-position of theterminal indole heterocycle substituted by a N-ω-alkyl sulfonic acidfunction. In these publications, cyanine compounds having more than twosulfonic acid function substituents exhibited higher solubility andcorrespondingly a lower tendency to dimer formation, in comparison tocyanine compounds (Cy3, Cy5) described in U.S. Pat. No. 5,627,027.

The disclosed cyanine compounds are useful as labels in optical,especially fluorescence optical, determination and detection methods.The compounds have high hydrophilicity, high molar absorbance, highphoto-stability, and high storage stability. These compounds can beexcited by monochromatic (e.g., lasers, laser diodes) or polychromatic(e.g., white light sources) light in the ultraviolet (UV), visible, andnear infrared (NIR) spectral region to generate emission of fluorescencelight.

Typical application methods are based on the reaction of the compoundswith biomolecules such as proteins (e.g., antigens, antibodies, etc.),DNA and/or RNA segments, etc. with the corresponding complimentaryspecies. Thus, among other embodiments, the compounds are useful tomeasure enzyme kinetics, receptor-ligand interactions, and nucleic acidhybridization kinetics in vitro and/or in vivo. The compounds are usefulfor the pharmacological characterization of receptors and/or drugs.Applications include, but are not limited to, uses in medicine,pharmacy, biological sciences, materials sciences, environmentalcontrol, detection of organic and inorganic micro samples occurring innature, etc.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 schematically shows conjugation between an azide-modifiedmolecule and a fluorophore.

FIG. 2 schematically shows another conjugation between an azide-modifiedmolecule and a fluorophore.

FIG. 3 schematically shows another conjugation between aphosphine-activated compound and an azide-labeled molecule.

FIG. 4 shows one embodiment of a heterobifunctional linking moiety.

FIG. 5 shows absorbance of various phosphine-containing compounds.

FIG. 6 shows relative fluorescence units (RFU) for variousphosphine-containing compounds.

FIG. 7 shows RFU for various phosphine-containing compounds.

FIG. 8 shows absorbance of conjugates comprising variousphosphine-containing compounds.

FIG. 9 shows dye to protein ratio (D/P) for conjugates comprisingvarious phosphine-containing compounds.

FIG. 10 shows D/P for conjugates comprising various phosphine-containingcompounds.

FIG. 11 shows relative RFU for conjugates comprising variousphosphine-containing compounds.

FIG. 12 shows RFU for conjugates comprising various phosphine-containingcompounds.

FIG. 13 shows fluorescence intensity for conjugates comprising variousphosphine-containing compounds.

FIG. 14 shows fluorescence intensity for conjugates comprising variousphosphine-containing compounds.

FIG. 15 shows sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) for conjugates comprising various phosphine-containingcompounds.

FIG. 16 shows SDS-PAGE for conjugates comprising variousphosphine-containing compounds.

FIG. 17 shows SDS-PAGE for conjugates comprising variousphosphine-containing compounds.

FIG. 18 shows SDS-PAGE for conjugates comprising variousphosphine-containing compounds.

FIG. 19 shows fluorescence of conjugates comprising variousphosphine-containing compounds in a gel.

FIG. 20 shows fluorescence images of SDS-PAGE gels of conjugatescomprising various phosphine-containing compounds.

FIG. 21 shows fluorescence images of SDS-PAGE gels of conjugatescomprising various phosphine-containing compounds.

FIG. 22 shows fluorescence intensity for conjugates comprising variousphosphine-containing compounds in a gel.

FIG. 23 shows conjugates comprising various phosphine-containingcompounds in a gel.

FIG. 24 shows conjugates comprising various phosphine-containingcompounds in a gel.

FIG. 25 shows fluorescence intensity for conjugates comprising variousphosphine-containing compounds in a gel.

FIG. 26 shows RFU for conjugates comprising various phosphine-containingcompounds.

FIG. 27 shows RFU for conjugates comprising various phosphine-containingcompounds.

FIG. 28 shows fluorescence images of cells labeled with variousphosphine-containing compounds.

FIG. 29A shows Spot Average Intensity of images of FIG. 28.

FIG. 29B shows Spot Average Intensity of images of FIG. 28.

FIG. 30 shows Spot Fluorescence Intensity of the live cell images ofFIG. 28.

FIG. 31 shows Spot Fluorescence Intensity of the fixed cell images ofFIG. 28.

FIG. 32 shows fluorescence images of cells labeled with variousphosphine-containing compounds.

FIG. 33A shows Spot Average Intensity of images of FIG. 32.

FIG. 33B shows Spot Average Intensity of images of FIG. 32.

FIG. 34 shows Spot Fluorescence Intensity of the live cell images ofFIG. 32.

FIG. 35 shows Spot Fluorescence Intensity of the fixed cell images ofFIG. 32.

FIG. 36 shows fluorescence images of cells labeled with variousphosphine-containing compounds.

FIG. 37A shows Spot Average Intensity of images of FIG. 36.

FIG. 37B shows Spot Average Intensity of images of FIG. 36.

FIG. 38 shows Spot Fluorescence Intensity of the live cell images ofFIG. 36.

FIG. 39 shows Spot Fluorescence Intensity of the fixed cell images ofFIG. 36.

FIG. 40 shows fluorescence images of cells labeled with variousphosphine-containing compounds.

FIG. 41A shows Spot Average Intensity of images of FIG. 40.

FIG. 41B shows Spot Average Intensity of images of FIG. 40.

FIG. 42 shows Spot Fluorescence Intensity of the live cell images ofFIG. 40.

FIG. 43 shows Spot Fluorescence Intensity of the fixed cell images ofFIG. 40.

FIG. 44 shows fluorescence images of cells labeled with variousphosphine-containing compounds.

FIG. 45A shows Spot Average Intensity of images of FIG. 44.

FIG. 45B shows Spot Average Intensity of images of FIG. 44.

FIG. 46 shows Spot Fluorescence Intensity of the live cell images ofFIG. 44.

FIG. 47 shows Spot Fluorescence Intensity of the fixed cell images ofFIG. 44.

FIG. 48 shows fluorescence images of cells labeled with variousphosphine-containing compounds.

FIG. 49A shows Spot Average Intensity of images of FIG. 48.

FIG. 49B shows Spot Average Intensity of images of FIG. 48.

FIG. 50 shows Spot Fluorescence Intensity of the live cell images ofFIG. 48.

FIG. 51 shows Spot Fluorescence Intensity of the fixed cell images ofFIG. 48.

FIG. 52 shows fluorescence images of cells labeled with variousphosphine-containing compounds.

FIG. 53 shows Spot Fluorescence Intensity of the live cell images ofFIG. 52.

FIG. 54 shows Spot Fluorescence Intensity of the fixed cell images ofFIG. 52.

FIG. 55 shows fluorescence images of cells labeled with variousphosphine-containing compounds.

FIG. 56 shows Spot Fluorescence Intensity of the live cell images ofFIG. 55.

FIG. 57 shows Spot Fluorescence Intensity of the fixed cell images ofFIG. 55.

FIG. 58 shows fluorescence images of cells labeled with variousphosphine-containing compounds.

FIG. 59 shows Spot Fluorescence Intensity of the live cell images ofFIG. 58.

FIG. 60 shows Spot Fluorescence Intensity of the fixed cell images ofFIG. 58.

FIG. 61 shows SDS-PAGE of cell lysates labeled with variousphosphine-containing compounds.

FIG. 62 shows SDS-PAGE of cell lysates labeled with variousphosphine-containing compounds.

FIG. 63 shows band intensity of the gel image of FIG. 61.

FIG. 64 shows band intensity of the gel image of FIG. 62.

FIG. 65A shows a fluorescence image of a cell showing secretory vesiclelabeling with a phosphine-containing compound.

FIG. 65B shows a fluorescence image of a cell showing co-localization ofa secretory protein with the image of FIG. 65A.

FIG. 66A shows a fluorescence image of a cell showing secretory vesiclelabeling with a phosphine-containing compound.

FIG. 66B shows a fluorescence image of a cell showing co-localization ofa secretory protein with the image of FIG. 66A.

FIG. 67 shows protein detection on nitrocellulose (NC) using aphosphine-containing compound.

In one embodiment, the cyanine compounds have, in an N-position of oneheterocycle, an ethylene glycol group, and the other heterocycle has, ina N-position, a function for conjugating the compound to a biomolecule.

In one embodiment, the cyanine compounds have, in an N-position of oneheterocycle, an ethylene glycol polymer (i.e., poly(ethylene)glycolabbreviated as PEG), and the other heterocycle has, in a N-position, afunction for conjugating the compound to a biomolecule.

In one embodiment, the compound is according to general formula I

where each of R¹ and R² is the same or different and is independentlyselected from the group consisting of an aliphatic, heteroaliphatic,sulfoalkyl group, or heteroaliphatic with terminal SO₃; X is selectedfrom the group consisting of —OH, —SH, —NH₂, —NH—NH₂, —F, —Cl, —Br, I,—NHS (hydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O-STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, and—NR-L-NH—CO—CH2-I, where R is —H or an aliphatic or heteroaliphaticgroup, and L is selected from the group consisting of a divalent linear,crossed, or cyclic alkyl group optionally substituted by at least oneoxygen atom and/or sulfur atom; Kat is a number of Na⁺, K⁺, Ca²⁺,ammonia, or other cation(s) needed to compensate the negative chargebrought by the cyanine; m is an integer from 0 to 5 inclusive; o is aninteger from 0 to 12 inclusive; and n is an integer from 1 to 3inclusive. The benzocyanine form of the above compound is

where the substituents are as described above.

In one embodiment, the compound is according to general formula I, whereeach of R1 is R2 is sulfoalkyl; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 1. In one embodiment, thecompound is according to general formula I, where each of R1 is R2 issulfoalkyl; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3;and n is 1. In one embodiment, the compound is according to generalformula I, where each of R1 is R2 is sulfoalkyl; X is —OH, —NHS, —O-TFP,or —NR-L-maleimide; m is 2; o is 3; and n is 1. In one embodiment, thecompound is according to general formula I, where each of R1 is R2 issulfoalkyl; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3;and n is 1. In one embodiment, the compound is according to generalformula I, where each of R1 is R2 is sulfoalkyl; X is —OH, —NHS, —O-TFP,or —NR-L-maleimide; m is 4; o is 3; and n is 1. In one embodiment, thecompound is according to general formula I, where each of R1 is R2 issulfoalkyl; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3;and n is 1.

In one embodiment, the compound is according to general formula I, whereeach of R1 is R2 is sulfoalkyl; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 2. In one embodiment, thecompound is according to general formula I, where each of R1 and R2 issulfoalkyl; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3;and n is 2. In one embodiment, the compound is according to generalformula I, where each of R1 and R2 is sulfoalkyl; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 2. In oneembodiment, the compound is according to general formula I, where eachof R1 and R2 is sulfoalkyl; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide;m is 3; o is 3; and n is 2. In one embodiment, the compound is accordingto general formula I, where each of R1 and R2 is sulfoalkyl; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 2. In oneembodiment, the compound is according to general formula I, where eachof R1 and R2 is sulfoalkyl; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide;m is 5; o is 3; and n is 2.

In one embodiment, the compound is according to general formula I, whereeach of R1 and R2 is sulfoalkyl; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 3. In one embodiment, thecompound is according to general formula I, where each of R1 and R2 issulfoalkyl; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3;and n is 3. In one embodiment, the compound is according to generalformula I, where each of R1 and R2 is sulfoalkyl; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 3. In oneembodiment, the compound is according to general formula I, where eachof R1 and R2 is sulfoalkyl; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide;m is 3; o is 3; and n is 3. In one embodiment, the compound is accordingto general formula I, where each of R1 and R2 is sulfoalkyl; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 3. In oneembodiment, the compound is according to general formula I, where eachof R1 and R2 is sulfoalkyl; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide;m is 5; o is 3; and n is 3.

In one embodiment, the compound has general formula II

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl group, or heteroaliphatic with terminal SO₃;each of R⁷ and R⁸ is the same or different and is independently selectedfrom either H, SO₃, sulfoalkyl, heteroaliphatic, or heteroaliphatic withterminal SO₃; X is selected from the group consisting of —OH, —SH, —NH₂,—NH—NH₂, —F, —Cl, —Br, I, —NHS (hydroxysuccinimidyl/sulfosuccinimidyl),—O-TFP (2,3,5,6-tetrafluorophenoxy), —O-STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, and—NR-L-NH—CO—CH2-I, where R is —H or an aliphatic or heteroaliphaticgroup, and L is selected from the group consisting of a divalent linear,crossed, or cyclic alkyl group optionally substituted by at least oneoxygen atom and/or sulfur atom; Kat is a number of Na⁺, K⁺, Ca²⁺,ammonia, or other cation(s) needed to compensate the negative chargebrought by the cyanine; m is an integer from 0 to 5 inclusive; o is aninteger from 0 to 12 inclusive; and n is an integer from 1 to 3inclusive. The benzocyanine form of the above compound is

as described above, where each R is selected from the group describedfor R7 and R8.

In one embodiment, the compound has general formula III

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl group, or heteroaliphatic with terminal SO₃;each of R⁷ and R⁸ is the same or different and is independently selectedfrom either H, SO₃, sulfoalkyl, heteroaliphatic, or heteroaliphatic withterminal SO₃; each of R9 and R10 is the same or different and isindependently selected from either aryl groups, substituted aryl groups,or cycloalkyl groups; R11 is an electrophilic group selected from thegroup consisting of a carboxylic acid, an alkyl ester, an aryl ester, asubstituted aryl ester, an aldehyde, and amide, an aryl amide, an alkylhalide, a thioester, a sulfonyl ester, and alkyl ketone, an aryl ketone,a substituted aryl ketone, a halosulfonyl, a nitrile, and a nitro; L isa linking group that forms a covalent bond between the dye and thephosphine; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s)needed to compensate the negative charge brought by the cyanine; m is aninteger from 0 to 5 inclusive; o is an integer from 0 to 12 inclusive;and n is an integer from 1 to 3 inclusive. The benzocyanine form of theabove compound is

as described above, where each R is selected from the group describedfor R7 and R8.

In one embodiment, the compound is according to general formula III,where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl; eachof R7 and R8 is SO₃; m is 0; o is 3; and n is 1. In one embodiment, thecompound is according to general formula III, where each of R1 and R2 issulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃; m is1; o is 3; and n is 1. In one embodiment, the compound is according togeneral formula III, where each of R1 and R2 is sulfoalkyl; each of R5and R6 is methyl; each of R7 and R8 is SO₃; m is 2; o is 3; and n is 1.In one embodiment, the compound is according to general formula III,where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl; eachof R7 and R8 is SO₃; m is 3; o is 3; and n is 1. In one embodiment, thecompound is according to general formula III, where each of R1 and R2 issulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃; m is4; o is 3; and n is 1. In one embodiment, the compound is according togeneral formula III, where each of R1 and R2 is sulfoalkyl; each of R5and R6 is methyl; each of R7 and R8 is SO₃; m is 5; o is 3; and n is 1.

In one embodiment, the compound is according to general formula III,where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl; eachof R7 and R8 is SO₃; m is 0; o is 3; and n is 2. In one embodiment, thecompound is according to general formula III, where each of R1 and R2 issulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃; m is1; o is 3; and n is 2. In one embodiment, the compound is according togeneral formula III, where each of R1 and R2 is sulfoalkyl; each of R5and R6 is methyl; each of R7 and R8 is SO₃; m is 2; o is 3; and n is 2.In one embodiment, the compound is according to general formula III,where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl; eachof R7 and R8 is SO₃; m is 3; o is 3; and n is 2. In one embodiment, thecompound is according to general formula III, where each of R1 and R2 issulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃; m is4; o is 3; and n is 2. In one embodiment, the compound is according togeneral formula III, where each of R1 and R2 is sulfoalkyl; each of R5and R6 is methyl; each of R7 and R8 is SO₃; m is 5; o is 3; and n is 2.

In one embodiment, the compound is according to general formula HI,where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl; eachof R7 and R8 is SO₃; m is 0; o is 3; and n is 3. In one embodiment, thecompound is according to general formula III, where each of R1 and R2 issulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃; m is1; o is 3; and n is 3. In one embodiment, the compound is according togeneral formula III, where each of R1 and R2 is sulfoalkyl; each of R5and R6 is methyl; each of R7 and R8 is SO₃; m is 2; o is 3; and n is 3.In one embodiment, the compound is according to general formula III,where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl; eachof R7 and R8 is SO₃; m is 3; o is 3; and n is 3. In one embodiment, thecompound is according to general formula III, where each of R1 and R2 issulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃; m is4; o is 3; and n is 3. In one embodiment, the compound is according togeneral formula III, where each of R1 and R2 is sulfoalkyl; each of R5and R6 is methyl; each of R7 and R8 is SO₃; m is 5; o is 3; and n is 3.

In one embodiment, the compound is according to general formula III,where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl; eachof R7 and R8 is SO₃; m is 0; o is 3; L is —CONH—CH₂—CH₂—NHCO—; each ofR9 and R10 is phenyl; R11 is COOCH₃; and n is 1. In one embodiment, thecompound is according to general formula III, where each of R1 and R2 issulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃; m is1; o is 3; L is —CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11is COOCH₃; and n is 1. In one embodiment, the compound is according togeneral formula III, where each of R1 and R2 is sulfoalkyl; each of R5and R6 is methyl; each of R7 and R8 is SO₃; m is 2; o is 3; L is—CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11 is COOCH₃; and nis 1. In one embodiment, the compound is according to general formulaIII, where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl;each of R7 and R8 is SO₃; m is 3; o is 3; L is —CONH—CH₂—CH₂—NHCO—; eachof R9 and R10 is phenyl; R11 is COOCH₃; and n is 1. In one embodiment,the compound is according to general formula III, where each of R1 andR2 is sulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃;m is 4; o is 3; L is —CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl;R11 is COOCH₃; and n is 1. In one embodiment, the compound is accordingto general formula III, where each of R1 and R2 is sulfoalkyl; each ofR5 and R6 is methyl; each of R7 and R8 is SO₃; m is 5; o is 3; L is—CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11 is COOCH₃; and nis 1.

In one embodiment, the compound is according to general formula III,where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl; eachof R7 and R8 is SO₃; m is 0; o is 3; L is —CONH—CH₂—CH₂—NHCO—; each ofR9 and R10 is phenyl; R11 is COOCH₃; and n is 2. In one embodiment, thecompound is according to general formula III, where each of R1 and R2 issulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃; m is1; o is 3; L is —CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11is COOCH₃; and n is 2. In one embodiment, the compound is according togeneral formula III, where each of R1 and R2 is sulfoalkyl; each of R5and R6 is methyl; each of R7 and R8 is SO₃; m is 2; o is 3; L is—CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11 is COOCH₃; and nis 2. In one embodiment, the compound is according to general formulaIII, where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl;each of R7 and R8 is SO₃; m is 3; o is 3; L is —CONH—CH₂—CH₂—NHCO—; eachof R9 and R10 is phenyl; R11 is COOCH₃; and n is 2. In one embodiment,the compound is according to general formula III, where each of R1 andR2 is sulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃;m is 4; o is 3; L is —CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl;R11 is COOCH₃; and n is 2. In one embodiment, the compound is accordingto general formula III, where each of R1 and R2 is sulfoalkyl; each ofR5 and R6 is methyl; each of R7 and R8 is SO₃; m is 5; o is 3; L is—CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11 is COOCH₃; and nis 2.

In one embodiment, the compound is according to general formula III,where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl; eachof R7 and R8 is SO₃; m is 0; o is 3; L is —CONH—CH₂—CH₂—NHCO—; each ofR9 and R10 is phenyl; R11 is COOCH₃; and n is 3. In one embodiment, thecompound is according to general formula Ill, where each of R1 and R2 issulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃; m is1; o is 3; L is —CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11is COOCH₃; and n is 3. In one embodiment, the compound is according togeneral formula III, where each of R1 and R2 is sulfoalkyl; each of R5and R6 is methyl; each of R7 and R8 is SO₃; m is 2; o is 3; L is—CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11 is COOCH₃; and nis 3. In one embodiment, the compound is according to general formulaIII, where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl;each of R7 and R8 is SO₃; m is 3; o is 3; L is —CONH—CH₂—CH₂—NHCO—; eachof R9 and R10 is phenyl; R11 is COOCH₃; and n is 3. In one embodiment,the compound is according to general formula III, where each of R1 andR2 is sulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃;m is 4; o is 3; L is —CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl;R11 is COOCH₃; and n is 3. In one embodiment, the compound is accordingto general formula III, where each of R1 and R2 is sulfoalkyl; each ofR5 and R6 is methyl; each of R7 and R8 is SO₃; m is 5; o is 3; L is—CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11 is COOCH₃; and nis 3.

In one embodiment, the compound is according to general formula III,where each of R¹, R², R⁶, and R⁶ is the same or different and isindependently selected from an aliphatic, heteroaliphatic, sulfoalkylgroup, heteroaliphatic with terminal SO₃, a PEG group P-L¹-Z where P isselected from an ethylene glycol group, a diethylene glycol group, and apolyethylene glycol group, where the polyethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, a sulfonamidegroup -L¹-SO₂NH—P-L¹-Z, and a caboxamide group -L¹-CONH—P-L¹-Z; each ofR⁷ and R⁸, and each R in the benzocyanine form is the same or differentand is independently selected from H, SO₃, a PEG group P-L¹-Z where P isselected from an ethylene glycol group, a diethylene glycol group, and apolyethylene glycol group, where the polyethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, a sulfonamidegroup -L¹-SO₂NH—P-L¹-Z, and a caboxamide group -L¹-CONH—P-L¹-Z; where L¹is selected from a divalent linear (—(CH₂)_(o)—, o=0 to 15), branched,or cyclic alkane group that can be substituted by at least one atomselected from oxygen, substituted nitrogen, and/or sulfur; where Z isselected from H, CH₃, an alkyl group, and a heteroalkyl group; Kat is anumber of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s) needed tocompensate the negative charge brought by the cyanine; m is an integerfrom 0 to 5 inclusive; o is an integer from 0 to 12 inclusive; and n isan integer from 1 to 3 inclusive; with the proviso that at least one ofR¹, R², R⁵, R⁶, R⁷, and R⁸ contains a PEG group.

In one embodiment, the compound is 550 Compound 1-phosphine

550 Compound 1-phosphine(2-((1E,3E)-3-(1-(6-(2-(3-(diphenylphosphino)-4-(methoxycarbonyl)benzamido)ethylamino)-6-oxohexyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)prop-1-enyl)-1-(2-methoxyethyl)-3-methyl-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonate)contains an ethylene glycol on the indole N of the left heterocycle. Themethyl group on the ethylene glycol prevents the terminal —OH fromoxidation. Oxidation is known to occur, over time, on an unprotected PEGterminus. Adding a methyl ether provides this protection, and preventsreaction with electrophilic reactive groups.

In one embodiment, the compound is 550 Compound 2-phosphine

550 Compound 2-phosphine(2-((1E,3E)-3-(1-(6-(2-(3-(diphenylphosphino)-4-(methoxycarbonyl)benzamido)ethylamino)-6-oxohexyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)prop-1-enyl)-1-(2-(2-methoxyethoxyl)ethyl)-3-methyl-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonate)contains a diethylene glycol on the indole N of the left heterocycle.The methyl group on the diethylene glycol prevents the terminal —OH fromoxidation. Oxidation is known to occur, over time, on an unprotected PEGterminus. Adding a methyl ether provides this protection, and preventsreaction with electrophilic reactive groups.

In one embodiment, the compound is 550 Compound 3-phosphine

550 Compound 3-phosphine(2-((1E,3E)-3-(1-(6-(2-(3-(diphenylphosphino)-4-(methoxycarbonyl)benzamido)ethylamino)-6-oxohexyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)prop-1-enyl)-1-(2-(2-(2-methoxyethoxyl)ethoxy)ethyl)-3-methyl-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonate)contains a polyethylene glycol on the indole N of the left heterocycle.The methyl group on the polyethylene glycol prevents the terminal —OHfrom oxidation. Oxidation is known to occur, over time, on anunprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.

In one embodiment, the compound is 550 Compound 4-phosphine

550 Compound 4-phosphine(2-((1E,3E)-3-(1-(6-(2-(3-(diphenylphosphino)-4-(methoxycarbonyl)benzamido)ethylamino)-6-oxohexyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)prop-1-enyl)-3-methyl-3-(3-sulfonatopropyl)-1-(2,5,8,11-tetraoxatridecan-13-yl)-3H-indolium-5-sulfonate)contains a polyethylene glycol on the indole N of the left heterocycle.

In one embodiment, the compound is 550 Compound 5-phosphine

550 Compound 5-phosphine(2-((1E,3E)-3-(1-(6-(2-(3-(diphenylphosphino)-4-(methoxycarbonyl)benzamido)ethylamino)-6-oxohexyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)prop-1-enyl)-1-(2,5,8,11,14-pentaoxahexadecan-16-yl)-3-methyl-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonate)contains a polyethylene glycol on the indole N of the left heterocycle.

In one embodiment, the compound is 550 Compound 6-phosphine

550 Compound 6-phosphine(2-((1E,3E)-3-(1-(6-(2-(3-(diphenylphosphino)-4-(methoxycarbonyl)benzamido)ethylamino)-6-oxohexyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)prop-1-enyl)-3-methyl-1-(2,5,8,11,14,17-hexaoxanonadecan-19-yl)-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonate)contains a polyethylene glycol on the indole N of the left heterocycle.

In embodiments, the degree of sulfonation is varied to, e.g., vary thecompound's degree of hydrophilicity or hydrophobicity. One non-limitingexample is a monosulfonate form of 550 Compound 1-phosphine, shownbelow, but it is understood that the single sulfo group can be at any ofthe described positions:

One non-limiting example is a disulfonate form of 550 Compound1-phosphine, shown below, but it is understood that each of the twosulfo groups can be at any of the described positions:

One non-limiting example is a trisulfonate form of 550 Compound1-phosphine, shown below, but it is understood that each of the threesulfo groups can be at any of the described positions:

One non-limiting example is a tetrasulfonate form of 550 Compound1-phosphine, shown below, but it is understood that each of the foursulfo groups can be at any of the described positions:

In one embodiment, the compound is 650 Compound 1-phosphine

650 Compound 1-phosphine(2-((1E,3E,5E)-5-(1-(6-(2-(3-(diphenylphosphino)-4-(methoxycarbonyl)benzamido)ethylamino)-6-oxohexyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)penta-1,3-dienyl)-1-(2-methoxyethyl)-3-methyl-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonate)contains an ethylene glycol on the indole N of the left heterocycle.

In one embodiment, the compound is 650 Compound 2-phosphine

650 Compound 2-phosphine(2-((1E,3E,5E)-5-(1-(6-(2-(3-(diphenylphosphino)-4-(methoxycarbonyl)benzamido)ethylamino)-6-oxohexyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)penta-1,3-dienyl)-1-(2-(2-methoxyethoxyl)ethyl)-3-methyl-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonate)contains a diethylene glycol on the indole N of the left heterocycle.

In one embodiment, the compound is 650 Compound 3-phosphine

650 Compound 3-phosphine(2-((1E,3E,5E)-5-(1-(6-(2-(3-(diphenylphosphino)-4-(methoxycarbonyl)benzamido)ethylamino)-6-oxohexyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)penta-1,3-dienyl)-1-(2-(2-(2-methoxyethoxyl)ethoxy)ethyl)-3-methyl-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonate)contains a polyethylene glycol on the indole N of the left heterocycle.

In one embodiment, the compound is 650 Compound 4-phosphine

650 Compound 4-phosphine(2-((1E,3E,5E)-5-(1-(6-(2-(3-(diphenylphosphino)-4-(methoxycarbonyl)benzamido)ethylamino)-6-oxohexyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)penta-1,3-dienyl)-3-methyl-3-(3-sulfonatopropyl)-1-(2,5,8,11-tetraoxatridecan-13-yl)-3H-indolium-5-sulfonate)contains a polyethylene glycol on the indole N of the left heterocycle.

In one embodiment, the compound is 650 Compound 5-phosphine

650 Compound 5-phosphine(2-((1E,3E,5E)-5-(1-(6-(2-(3-(diphenylphosphino)-4-(methoxycarbonyl)benzamido)ethylamino)-6-oxohexyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)penta-1,3-dienyl)-1-(2,5,8,11,14-pentaoxahexadecan-16-yl)-3-methyl-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonate)contains a polyethylene glycol on the indole N of the left heterocycle.

In one embodiment, the compound is 650 Compound 6-phosphine

650 Compound 6-phosphine(2-((1E,3E,5E)-5-(1-(6-(2-(3-(diphenylphosphino)-4-(methoxycarbonyl)benzamido)ethylamino)-6-oxohexyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)penta-1,3-dienyl)-3-methyl-1-(2,5,8,11,14,17-hexaoxanonadecan-19-yl)-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonate)contains a polyethylene glycol on the indole N of the left heterocycle.

In embodiments, the degree of sulfonation is varied to, e.g., vary thecompound's degree of hydrophilicity or hydrophobicity. One non-limitingexample is a monosulfonate form of 650 Compound 1-phosphine, shownbelow, but it is understood that the single sulfo group can be at any ofthe described positions:

One non-limiting example is a disulfonate form of 650 Compound1-phosphine, shown below, but it is understood that each of the twosulfo groups can be at any of the described positions:

One non-limiting example is a trisulfonate form of 650 Compound1-phosphine, shown below, but it is understood that each of the threesulfo groups can be at any of the described positions:

One non-limiting example is a tetrasulfonate form of 650 Compound1-phosphine, shown below, but it is understood that each of the foursulfo groups can be at any of the described positions:

In one embodiment, the compound is 755 Compound 1-phosphine

755 Compound 1-phosphine(2-((1E,3E,5E,7E)-7-(1-(6-(2-(3-(diphenylphosphino)-4-(methoxycarbonyl)benzamido)ethylamino)-6-oxohexyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)hepta-1,3,5-trienyl)-1-(2-methoxyethyl)-3-methyl-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonate)contains an ethylene glycol on the indole N of the left heterocycle.

In one embodiment, the compound is 755 Compound 2-phosphine

755 Compound 2-phosphine(2-((1E,3E,5E,7E)-7-(1-(6-(2-(3-(diphenylphosphino)-4-(methoxycarbonyl)benzamido)ethylamino)-6-oxohexyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)hepta-1,3,5-trienyl)-1-(2-(2-methoxyethoxyl)ethyl)-3-methyl-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonate)contains a diethylene glycol on the indole N of the left heterocycle.

In one embodiment, the compound is 755 Compound 3-phosphine

755 Compound 3-phosphine(2-((1E,3E,5E,7E)-7-(1-(6-(2-(3-(diphenylphosphino)-4-(methoxycarbonyl)benzamido)ethylamino)-6-oxohexyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)hepta-1,3,5-trienyl)-1-(2-(2-(2-methoxyethoxyl)ethoxy)ethyl)-3-methyl-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonate)contains a polyethylene glycol on the indole N of the left heterocycle.

In one embodiment, the compound is 755 Compound 4-phosphine

755 Compound 4-phosphine(2-((1E,3E,5E,7E)-7-(1-(6-(2-(3-(diphenylphosphino)-4-(methoxycarbonyl)benzamido)ethylamino)-6-oxohexyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)hepta-1,3,5-trienyl)-3-methyl-3-(3-sulfonatopropyl)-1-(2,5,8,11-tetraoxatridecan-13-yl)-3H-indolium-5-sulfonate)contains a polyethylene glycol on the indole N of the left heterocycle.

In one embodiment, the compound is 755 Compound 5-phosphine

755 Compound 5-phosphine(2-((1E,3E,5E,7E)-7-(1-(6-(2-(3-(diphenylphosphino)-4-(methoxycarbonyl)benzamido)ethylamino)-6-oxohexyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)hepta-1,3,5-trienyl)-1-(2,5,8,11,14-pentaoxahexadecan-16-yl)-3-methyl-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonate)contains a polyethylene glycol on the indole N of the left heterocycle.

In one embodiment, the compound is 755 Compound 6-phosphine

755 Compound 6-phosphine(2-((1E,3E,5E,7E)-7-(1-(6-(2-(3-(diphenylphosphino)-4-(methoxycarbonyl)benzamido)ethylamino)-6-oxohexyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)hepta-1,3,5-trienyl)-3-methyl-1-(2,5,8,11,14,17-hexaoxanonadecan-19-yl)-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonate)contains a polyethylene glycol on the indole N of the left heterocycle.

In embodiments, the degree of sulfonation is varied to, e.g., vary thecompound's degree of hydrophilicity or hydrophobicity. One non-limitingexample is a monosulfonate form of 755 Compound 1-phosphine, shownbelow, but it is understood that the single sulfo group can be at any ofthe described positions:

One non-limiting example is a disulfonate form of 755 Compound1-phosphine, shown below, but it is understood that the each of the twosulfo groups can be at any of the described positions:

One non-limiting example is a trisulfonate form of 755 Compound1-phosphine, shown below, but it is understood that the each of thethree sulfo groups can be at any of the described positions:

One non-limiting example is a tetrasulfonate form of 755 Compound1-phosphine, shown below, but it is understood that the each of the foursulfo groups can be at any of the described positions:

In one embodiment, the compound has general formula IV

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl group, or heteroaliphatic with terminal SO₃;each of R⁷ and R⁸ is the same or different and is independently selectedfrom either H, SO₃, sulfoalkyl, heteroaliphatic, or heteroaliphatic withterminal SO₃; each of R9 and R10 is the same or different and isindependently selected from either aryl groups, substituted aryl groups,or cycloalkyl groups; R11 is an electrophilic group selected from thegroup consisting of a carboxylic acid, an alkyl ester, an aryl ester, asubstituted aryl ester, an aldehyde, and amide, an aryl amide, an alkylhalide, a thioester, a sulfonyl ester, and alkyl ketone, an aryl ketone,a substituted aryl ketone, a halosulfonyl, a nitrile, and a nitro; L isa linking group that forms a covalent bond between the dye and thephosphine; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s)needed to compensate the negative charge brought by the cyanine; m is aninteger from 0 to 5 inclusive; p is an integer from 1 to 6 inclusive;and n is an integer from 1 to 3 inclusive. The benzocyanine form of theabove compound is

as described above, where each R is selected from the group describedfor R7 and R8.

In one embodiment, the compound is according to general formula IV,where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl; eachof R7 and R8 is SO₃; m is 0; p is 1; L is —CONH—CH₂—CH₂—NHCO—; each ofR9 and R10 is phenyl; R11 is COOCH₃; and n is 1. In one embodiment, thecompound is according to general formula IV, where each of R1 and R2 issulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃; m is0; p is 2; L is —CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11is COOCH₃; and n is 1. In one embodiment, the compound is according togeneral formula IV, where each of R1 and R2 is sulfoalkyl; each of R5and R6 is methyl; each of R7 and R8 is SO₃; m is 0; p is 3; L is—CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11 is COOCH₃; and nis 1. In one embodiment, the compound is according to general formulaIV, where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl;each of R7 and R8 is SO₃; m is 0; p is 4; L is —CONH—CH₂—CH₂—NHCO—; eachof R9 and R10 is phenyl; R11 is COOCH₃; and n is 1. In one embodiment,the compound is according to general formula IV, where each of R1 and R2is sulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃; mis 0; p is 5; L is —CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl;R11 is COOCH₃; and n is 1. In one embodiment, the compound is accordingto general formula IV, where each of R1 and R2 is sulfoalkyl; each of R5and R6 is methyl; each of R7 and R8 is SO₃; m is 0; p is 6; L is—CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11 is COOCH₃; and nis 1.

In one embodiment, the compound is according to general formula IV,where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl; eachof R7 and R8 is SO₃; m is 0; p is 1; L is —CONH—CH₂—CH₂—NHCO—; each ofR9 and R10 is phenyl; R11 is COOCH₃; and n is 2. In one embodiment, thecompound is according to general formula IV, where each of R1 and R2 aresulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 are SO₃; m is0; p is 2; L is —CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11is COOCH₃; and n is 2. In one embodiment, the compound is according togeneral formula IV, where each of R1 and R2 are sulfoalkyl; each of R5and R6 is methyl; each of R7 and R8 are SO₃; m is 0; p is 3; L is—CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11 is COOCH₃; and nis 2. In one embodiment, the compound is according to general formulaIV, where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl;each of R7 and R8 is SO₃; m is 0; p is 4; L is —CONH—CH₂—CH₂—NHCO—; eachof R9 and R10 is phenyl; R11 is COOCH₃; and n is 2. In one embodiment,the compound is according to general formula IV, where each of R1 and R2is sulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃; mis 0; p is 5; L is —CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl;R11 is COOCH₃; and n is 2. In one embodiment, the compound is accordingto general formula IV, where each of R1 and R2 is sulfoalkyl; each of R5and R6 is methyl; each of R7 and R8 is SO₃; m is 0; p is 6; L is—CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11 is COOCH₃; and nis 2.

In one embodiment, the compound is according to general formula IV,where each of R1 and R2 are sulfoalkyl; each of R5 and R6 are methyl;each of R7 and R8 are SO₃; m is 0; p is 1; L is —CONH—CH₂—CH₂—NHCO—;each of R9 and R10 is phenyl; R11 is COOCH₃; and n is 3. In oneembodiment, the compound is according to general formula IV, where eachof R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl; each of R7 andR8 is SO₃; m is 0; p is 2; L is —CONH—CH₂—CH₂—NHCO—; each of R9 and R10is phenyl; R11 is COOCH₃; and n is 3. In one embodiment, the compound isaccording to general formula IV, where each of R1 and R2 is sulfoalkyl;each of R5 and R6 is methyl; each of R7 and R8 is SO₃; m is 0; p is 3; Lis —CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11 is COOCH₃; andn is 3. In one embodiment, the compound is according to general formulaIV, where each of R1 and R2 sulfoalkyl; each of R5 and R6 is methyl;each of R7 and R8 is SO₃; m is 0; p is 4; L is —CONH—CH₂—CH₂—NHCO—; eachof R9 and R10 is phenyl; R11 is COOCH₃; and n is 3. In one embodiment,the compound is according to general formula IV, where each of R1 and R2is sulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃; mis 0; p is 5; L is —CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl;R11 is COOCH₃; and n is 3. In one embodiment, the compound is accordingto general formula III, where each of R1 and R2 is sulfoalkyl; each ofR5 and R6 is methyl; each of R7 and R8 is SO₃; m is 0; p is 6; L is—CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11 is COOCH₃; and nis 3.

In one embodiment, the compound is according to general formula IV,where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl; eachof R7 and R8 is SO₃; m is 0; p is 1; L is —CONH—CH₂—CH₂—NHCO—; each ofR9 and R10 is phenyl; R11 is COOCH₃; and n is 1. In one embodiment, thecompound is according to general formula IV, where each of R1 and R2sulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃; m is1; p is 1; L is —CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11is COOCH₃; and n is 1. In one embodiment, the compound is according togeneral formula IV, where each of R1 and R2 is sulfoalkyl; each of R5and R6 is methyl; each of R7 and R8 is SO₃; m is 2; p is 1; L is—CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11 is COOCH₃; and nis 1. In one embodiment, the compound is according to general formulaIV, where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl;each of R7 and R8 is SO₃; m is 3; p is 1; L is —CONH—CH₂—CH₂—NHCO—; eachof R9 and R10 is phenyl; R11 is COOCH₃; and n is 1. In one embodiment,the compound is according to general formula IV, where each of R1 and R2is sulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃; mis 4; p is 1; L is —CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl;R11 is COOCH₃; and n is 1. In one embodiment, the compound is accordingto general formula IV, where each of R1 and R2 is sulfoalkyl; each of R5and R6 is methyl; each of R7 and R8 is SO₃; m is 5; p is 1; L is—CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11 is COOCH₃; and nis 1.

In one embodiment, the compound is according to general formula IV,where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl; eachof R7 and R8 is SO₃; m is 0; p is 1; L is —CONH—CH₂—CH₂—NHCO—; each ofR9 and R10 is phenyl; R11 is COOCH₃; and n is 2. In one embodiment, thecompound is according to general formula IV, where each of R1 and R2 issulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃; m is1; p is 1; L is —CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11is COOCH₃; and n is 2. In one embodiment, the compound is according togeneral formula IV, where each of R1 and R2 is sulfoalkyl; each of R5and R6 is methyl; each of R7 and R8 is SO₃; m is 2; p is 1; L is—CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11 is COOCH₃; and nis 2. In one embodiment, the compound is according to general formulaIV, where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl;each of R7 and R8 is SO₃; m is 3; p is 1; L is —CONH—CH₂—CH₂—NHCO—; eachof R9 and R10 is phenyl; R11 is COOCH₃; and n is 2. In one embodiment,the compound is according to general formula IV, where each of R1 and R2is sulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃; mis 4; p is 1; L is —CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl;R11 is COOCH₃; and n is 2. In one embodiment, the compound is accordingto general formula IV, where each of R1 and R2 is sulfoalkyl; each of R5and R6 is methyl; each of R7 and R8 is SO₃; m is 5; p is 1; L is—CONH—CH₂—CH₂—NHCO—; each of R9 is R10 are phenyl; R11 is COOCH₃; and nis 2.

In one embodiment, the compound is according to general formula IV,where each of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl; eachof R7 and R8 is SO₃; m is 0; p is 1; L is —CONH—CH₂—CH₂—NHCO—; each ofR9 and R10 is phenyl; R11 is COOCH₃; and n is 3. In one embodiment, thecompound is according to general formula IV, where each of R1 and R2 issulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃; m is1; p is 1; L is —CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11is COOCH₃; and n is 3. In one embodiment, the compound is according togeneral formula IV, where each of R1 and R2 is sulfoalkyl; each of R5and R6 is methyl; each of R7 and R8 is SO₃; m is 2; p is 1; L is—CONH—CH₂—CH₂—NHCO—; R9 and R10 is phenyl; R11 is COOCH₃; and n is 3. Inone embodiment, the compound is according to general formula IV, whereeach of R1 and R2 is sulfoalkyl; each of R5 and R6 is methyl; each of R7and R8 is SO₃; m is 3; p is 1; L is —CONH—CH₂—CH₂—NHCO—; each of R9 andR10 is phenyl; R11 is COOCH₃; and n is 3. In one embodiment, thecompound is according to general formula IV, where each of R1 and R2 issulfoalkyl; each of R5 and R6 is methyl; each of R7 and R8 is SO₃; m is4; p is 1; L is —CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11is COOCH₃; and n is 3. In one embodiment, the compound is according togeneral formula III, where each of R1 and R2 is sulfoalkyl; each of R5and R6 is methyl; each of R7 and R8 is SO₃; m is 5; p is 1; L is—CONH—CH₂—CH₂—NHCO—; each of R9 and R10 is phenyl; R11 is COOCH₃; and nis 3.

In one embodiment, the compound is according to general formula IV,where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from an aliphatic, heteroaliphatic, sulfoalkylgroup, heteroaliphatic with terminal SO₃, a PEG group P-L¹-Z where P isselected from an ethylene glycol group, a diethylene glycol group, and apolyethylene glycol group, where the polyethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, a sulfonamidegroup —C—SO₂NH—P-L¹-Z, and a caboxamide group -L¹-CONH—P-L¹-Z; each ofR⁷ and R⁸ and each R in the benzocyanine form is the same or differentand is independently selected from H, SO₃, a PEG group P-L¹-Z where P isselected from an ethylene glycol group, a diethylene glycol group, and apolyethylene glycol group, where the polyethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, a sulfonamidegroup -L¹-SO₂NH—P-L¹-Z, and a caboxamide group -L¹-CONH—P-L¹-Z; where L¹is selected from a divalent linear (—(CH₂)_(o)—, o=0 to 15), branched,or cyclic alkane group that can be substituted by at least one atomselected from oxygen, substituted nitrogen, and/or sulfur; where Z isselected from H, CH₃, an alkyl group, and a heteroalkyl group; Kat is anumber of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s) needed tocompensate the negative charge brought by the cyanine; m is an integerfrom 0 to 5 inclusive; p is an integer from 1 to 6 inclusive; and n isan integer from 1 to 3 inclusive; with the proviso that at least one ofR¹, R², R⁵, R⁶, R⁷, and R⁸ contains a PEG group.

In various embodiments, an ethylene glycol group, diethylene glycolgroup, and/or a polyethylene glycol group, which will collectively bereferred to as a PEG group, unless specifically defined, may be presentat position(s) in addition to such groups being present on the N atom(s)of the indole structure.

In embodiments, the degree of sulfonation is varied to, e.g., vary thecompound's degree of hydrophilicity or hydrophobicity. In embodiments,e.g., for functional assays, the inventive compounds are activated.Activation of the compound adds a chemical moiety such that the compoundis in a form that can be conjugated to a biological moiety. In oneexample, a phosphine group, as shown in general formulas III and IV, ispresent on the inventive compounds. In embodiments, the compound maycontain one or more substitutions of the polymethine linker.

The disclosed compounds are useful as chromophores and/or fluorophores.For example, they can be used for optical labelling and, therefore, forthe qualitative and/or quantitative detection of proteins, nucleicacids, oligomers, DNA, RNA, biological cells, lipids, mono-, oligo- andpolysaccharides, ligands, receptors, polymers, drugs, polymeric beads,etc.

The compounds containing the disclosed functionality or functionalitiesare synthesized using methods known in the art and as subsequentlydescribed.

Konig U.S. Pat. No. 1,524,791 and GB 434875 describes the coreindocyanine without additional functionalilities, its synthesis, and 3-,5-, and 7-membered polymethine chains.

Synthesis of numerous modifications of the core indocyanine structurehave been described. Such modifications provided variousfunctionalilities, e.g., synthesis of N-isothiocyanato-alkyl- andaromatic-carboxyalkyl-functionalized indocyanines, are described in U.S.Pat. Nos. 5,627,027; 6,048,982; 4,981,977; U.S. Publication No.2006/0199949; MANK, A. J. G. et al., Visible Diode Laser-InducedFluorescence Detection in Liquid Chromatography after PrecolumnDerivatization of Amines. Anal. Chem. vol. 67, pp. 1742-1748, 1995.

Synthesis of indocyanines with one or two N-carboxyalkyl functionalitieswere described in U.S. Pat. Nos. 5,268,486; 5,486,616; 5,569,587;5,569,766, and JP 03217837.

Synthesis of indocyanines containing C-carboxyalkyl groups weredescribed in JP 05-313304; U.S. Publication Nos. 2006/0099638,2006/0004188; 2002/0077487; 2002/0064794; and U.S. Pat. Nos. 6,977,305and 6,974,873.

Synthesis of indocyanines with N- and C-sulfoalkyl groups were describedin JP 05-313304; WO 2005/044923; and U.S. Publication No. 2007/0203343.

Synthesis of indocyanines with mixed C-carboxyalkyl and C-sulfoalkylwere described in EP 1792949 and U.S. Pat. No. 7,745,640.

Synthesis of indocyanaines having a PEG-containing N-carboxyalkyl spacerwere described in U.S. Pat. No. 6,939,532.

Functionalization of the N-carboxyalkyl with an amino-functionalizedPEG-alkyl chain, and N- and C-substituted PEG-alkyl chains, weredescribed in U.S. Publication No. 2009/0305410.

Synthesis of various polymethine bridge substitutions, and otherindocyanine functionalizations, are disclosed in Example 1-18, inStrekowski, Heterocyclic Polymethine Dyes: Synthesis, Properties andApplications, (2008) Springer-Verlag, Berlin Heidelberg; Gragg,“Synthesis of Near-Infrared Heptamethine Cyanine Dyes” (2010) ChemistryTheses. Paper 28; Patonay et al. (2004) Noncovalent Labeling ofBiomolecules with Red and Near-Infrared Dyes. Molecules 9 (2004) 40-49;and U.S. Pat. No. 7,172,907.

In one embodiment, the compound is synthesized by a condensationreaction, known to one skilled in the art, of the two differentlysubstituted indole heterocycles separated by a (poly)methine linker orbridge, e.g., C1, C3, or C5. Other synthesis methods are possible. Asonly one example, one of the indole heterocycles is first reacted withthe C1, C3, or C5 linker. The 1:1 condensation product is isolated, andthen condensed with the second indole heterocycle to result in thecyanine compound. The sequence of reacting the indole heterocycles isirrelevant. Thus, a plurality of differently functionalized, stronglyhydrophilic, diastereomeric compounds that differ in total charge andspecificity/reactivity of the active groups used for theirimmobilization, were prepared.

Conjugates of the compounds are prepared by covalently coupling thecompounds to a biomolecule using the functional substituent on theN-position of the indole ring. This functional substituent is activatedby routine protein chemistry reaction methods known to one skilled inthe art. The activated compound may be converted to, without limitation,an N-hydroxysuccinimide (NHS)-ester, an acid fluoride, atetrafluorophenyl (TFP)- or sulfotetrafluorophenyl (STP)-ester, aniodoacetyl group, a maleimide, a hydrazide, a sulfonyl chloride, aphenylazide. Methods for preparing such compounds are known to oneskilled in the art. In one embodiment, the activated substituent is thenreacted with an amino group on the biomolecule under conditions to formthe linkage. In one embodiment, a non-activated carboxyl group on theN-position of the indole in the compound is coupled to an amine using acarbodimide.

Coupling between the compound and the biomolecule may be performed asfollows. The compound is reacted with the biomolecule in an organic oraqueous solution at a pH between pH 5 and pH 12, inclusive. The compoundneed not be dissolved in an organic solvent, such as dimethyl formamide(DMF) or dimethyl sulfoxide (DMSO) prior to adding the biomolecule. Inone embodiment, coupling reaction may be performed in a 100% aqueoussolution. In one embodiment, the coupling reaction occurs at roomtemperature (about 20° C. to about 22° C.).

To form a composition (dye), at least one biocompatible excipient isadded to the compound(s), as known to one of ordinary skill in the art.Excipients include but are not limited to buffers, solubility enhancingagents, stabilizing agents, etc.

In one embodiment, a kit for performing an assay method comprises adisclosed compound, and instructions for performing the method using thecompound.

The disclosed activated compounds (i.e., the compound modified with areactive group) are useful to label macromolecules (e.g., antibodies,streptavidin, etc) using methods known to one skilled in the art, e.g.,as disclosed in Hermanson, Bioconjugate Techniques, 2nd Ed., London,Elsevier Inc. 2008. The reaction is carried out for one hour to twohours at room temperature (about 20° C. to about 22° C.), and thendesalted by dialyzing against several changes of phosphate bufferedsaline (pH 7.2) or purified by gel filtration to remove the unreactedfluorescent dye. The resulting compound-biomolecule conjugate is usefulin applications such as detection of specific proteins in immunoassays,sugars in glycoproteins with lectins, protein-protein interactions,oligonucleotides in nucleic acid, hybridization, and in electrophoreticmobility shift assays (EMSA).

The resulting compound-biomolecule conjugates exhibit fluorescentproperties. They may be used in optical, including fluorescence optical,qualitative and quantitative determination methods. Examples of suchmethods include, but are not limited to, microscopy, immunoassays,hybridization methods, chromatographic and electrophoretic methods,fluorescence resonance energy transfer (FRET) systems, high throughputscreenings, analysis of receptor-ligand interactions on a microarray,etc.

Compounds of any of the embodiments can be used as dyes for opticallabelling of organic or inorganic biomolecules, also referred to asrecognition units. Recognition units are molecules having specificityand/or affinity for a specific group of molecules. Examples ofrecognition units include, but are not limited to, antibodies that haveaffinity for antigens, enzymes that bind and/or react with a specificbond or bonds within a sequence of amino acids in a peptide or reactwith a substrate, cofactors such as metals that enhance or inhibitspecific interactions, lectins that bind specific sugars or sugarsequences (e.g., oligosaccharides, polysaccharides, dextrans, etc.),biotin binding proteins such as avidin and streptavidin that bind biotinand biotinylated molecules, antibody binding proteins such as Protein A,Protein G, Protein A/G and Protein L, sequences of amino acids or metalsthat have affinity for each other (e.g., histidine sequences bind nickelor copper, phosphate containing proteins that bind gallium, aluminium,etc.), specific sequences of nucleic acids such as DNA and/or RNAoligonucleotides that have affinity for proteins, specific sequences ofamino acids that have affinity for DNA and/or RNA, haptens, carotenoids,hormones (e.g., neurohormone), neurotransmitters, growth factors,toxins, biological cells, lipids, receptor binding drugs or organic orinorganic polymeric carrier materials, fluorescent proteins such asphycobilliproteins (e.g., phycoethrin, allophycocyanin), etc. The ionicinteractions between these recognition units and the disclosed compoundsresults in labeling of the recognition units. The recognition unit andcompound can be covalently bound. The result is a conjugate forqualitative or quantitative determination of various biomaterials orother organic or inorganic materials using optical methods.

The inventive compounds and/or conjugates are useful in optical,including fluorescence optical, qualitative and/or quantitativedetermination methods to diagnose properties of cells (molecularimaging), in biosensors (point of care measurements), for investigationof the genome, and in miniaturizing technologies. Microscopy, cytometry,cell sorting, fluorescence correlation spectroscopy (FCS), ultra highthroughput screening (uHTS), multicolour fluorescence in situhybridisation (mc-FISH), FRET-systems and microarrays (DNA- and proteinchips) are exemplary application fields. As known to one skilled in theart, a microarray, a grid-like arrangement where more than two differentmolecules are immobilized in a known predefined region on at least onesurface, is useful to evaluate receptor ligand interactions. As known toone skilled in the art, a receptor is a naturally occurring or syntheticmolecule that exhibits an affinity to a given ligand. Receptors can beused in a pure form or bound to another specie. Receptors can be coupledcovalently or noncovalently to a binding partner either directly orindirectly (e.g., through a coupling mediator). Receptor examplesinclude, but are not limited to, agonists and antagonists for cellmembrane receptors, toxins and other poisons, viral epitopes, hormonelike opiates and steroids, hormone receptors, peptides, enzymes, enzymesubstrates, drugs acting as cofactors, lectins, sugars,oligonucleotides, nucleic acids, oligosaccharides, cells, cellfragments, tissue fragments, proteins, antibodies, etc. As known to oneskilled in the art, a ligand is a molecule that is recognized by acertain receptor. Ligand examples include, but are not limited to,agonists and antagonists for cell membrane receptors, toxins and otherpoisons, viral epitopes, hormones like opiates and steroids, hormonereceptors, peptides, enzymes, enzyme substrates, drugs acting ascofactors, lectins, sugars, oligonucleotides, nucleic acids,oligosaccharides, proteins, antibodies, etc.

In one embodiment, the inventive phosphine compounds and/or conjugatesare used as chemoselective, also termed bioorthogonal, chemistryreagents. Because azides are not naturally present in cells andphosphines do not naturally react with azides in cells, adding theinventive phosphines to cells specifically labels only azide-containingcell components with no non-specific labeling. Using metabolic labeling,azide groups, which are the reactive targets of the phosphine-activatedfluors, were introduced into proteins or other cellular targets usingazide reagents or through in vivo labeling with azide-derivatives ofnaturally occurring metabolic building blocks.

Metabolic labeling refers to methods in which chemical detection- oraffinity-tags are added to biomolecules in vivo using the endogenoussynthesis and modification machinery of live cells. Analogs of molecularbuilding blocks (e.g., amino acids, sugars) designed to containspecifically targetable tags that do not interfere with the cellularmetabolic machinery provide a mechanism for metabolic labeling,permitting a number of powerful experimental approaches forinvestigating cellular pathways. Metabolic labeling with bioorthogonalmonosaccharides (sugars) that are used by cells to glycosylate proteinsand other cell constituents provides a variety of experimentalapproaches. The effects of drugs or other treatment conditions on totalor sugar-specific glycosylation can be measured. When bioorthogonalazido-sugar derivatives are supplied to live cells, they areincorporated into glycoproteins by endogenous post-translationalmodification mechanisms. The azide-tagged molecules are then selectivelylabeled or conjugated to phosphine-activated molecules.

This specific cellular labeling with phosphines occurs in a two-stepprocess: (1) conjugating a cell protein, carbohydrate, etc. or otherbiomolecule with an azide group; then (2) adding the inventive phosphinecompound to the azide-containing biomolecule. Staudinger ligation(azide-phosphine) chemistry is a crosslinking technique amenable to invivo metabolic labeling. Because the azide component of thechemoselective reaction pair is small, it can be supplied to live cellsin the form of bioorthogonal molecules that substitute for cellularbuilding blocks used to synthesize proteins or other macromolecules.

Bioorthogonal chemistry refers to any chemical reaction that can occurinside living systems without interfering with native biochemicalprocesses. The concept of the bioorthogonal reactions have enabled realtime study of biomolecules such as glycans, proteins, and lipids inliving systems without cellular toxicity. Several chemical ligationstrategies have been developed that fulfil bioorthogonalityrequirements, e.g., the isonitrile-based CLICK-iT® reaction (FIG. 1);the 1,3-dipolar cycloaddition between azides and cyclooctynes, alsotermed copper-less Click Reaction (FIG. 2) using dibenzocyclooctyne; theStaudinger ligation (FIG. 3); and reaction between nitrones andcyclooctynes, oxime/hydrazone formation from aldehydes and ketones, thetetrazine ligation, and the quadricyclane ligation.

As stated previously, bioorthogonal chemical reactions typically proceedin two steps. First, a cellular substrate or target molecule is modifiedwith a bioorthogonal functional group, also termed a chemical reporter,and introduced to the cell; substrates include metabolites, enzymeinhibitors, antibodies, etc. The functional group is designed so it doesnot dramatically alter the structure of the substrate, thus notaffecting substrate bioactivity. Secondly, a probe containing acomplementary functional group is introduced to react with and label thesubstrate.

Whether a particular labeling chemistry can be used in metaboliclabeling depends upon its chemoselectivity (reaction specificity) andmetabolic compatibility (i.e., production of bioorthogonal derivativesthrough metabolism). In this chemoselective ligation strategy, onecomponent of the reaction pair is supplied as a substitute (an analog)of a naturally occurring molecule required for catabolism of the targetmacromolecules. Bioorthogonal means that the biological function of themolecule is unaffected by the reactive group it contains, i.e., thereactive group is invisible to the biological system. The azide group inthe Staudinger reaction pair has this bioorthogonal property. Whensupplied to cells, synthetic azide-containing analogs of amino acids orsugars are incorporated during protein synthesis or post-translationalglycosylation using cellular metabolic or regulatory machinery. Thus,the relevant chemoselective reactive group is added in vivo by metaboliclabeling. Alternatively, bioorthogonal derivatives can be incorporatedinto specific non-protein targets using in vitro enzymatic reactions.Once target molecules are labeled (tagged) with the bioorthogonal group(azide), they can be chemoselectively conjugated or tagged by reactionwith the desired phosphine-activated reagent (biotin, fluor, etc.) usingthe Staudinger reaction. Chemoselective ligation using bioorthogonalderivatives combines the simplicity of metabolically encoded tags withspecific labeling and the versatility of small-molecule probes.

The Staudinger ligation includes an ester group ortho to the phosphorusatom on one of the aryl rings to direct the aza-ylide to outcompeteimmediate hydrolysis by positioning the ester to increase localconcentration. The initial nucleophilic attack on the azide is therate-limiting step. The ylide reacts with the electrophilic ester trapthrough intramolecular cyclization to form a five-membered ring. Thisring undergoes hydrolysis to form a stable amide bond. The azide groupis particularly bioorthogonal because it is metabolically stable,extremely small rendering it favorable for cell permeability andavoiding perturbations, and not naturally existing in cells, thus havingno competing biological side reactions. Staudinger ligation requiresfewer harmful additives than other chemoselective ligation chemistriesdeveloped for use with biological samples. Although azide-alkyne(“click”) chemistry uses the same azide component as the azide-phosphine(Staudinger) chemistry, it requires special copper-containing reactionbuffers that have damaging effects on cellular components.

In one embodiment, the inventive phosphine-containing compounds areconjugated by bioorthogonal chemistry using the Staudinger ligation inwhich the inventive compound is conjugated with a biomolecule ofinterest, such as an antibody or a sugar moiety. In one embodiment, theconjugation reaction reacts a heterobifunctional linking moiety with abiomolecule of interest, resulting in a free azide group. The free azidegroup is then reacted with a phosphine on the compound to form abiomolecule-linker-phosphine compound conjugate. In one embodiment, theheterobifunctional linking moiety contains at one end a first group thatis reactive with an amine, such as an NHS ester, and at the other end asecond azide group, and a polyethylene glycol between the first andsecond groups. In one example, the polyethylene glycol is a PEG₄ group,shown in FIG. 4.

The inventive compounds are soluble; they readily dissolve inwater-miscible solvents (e.g., DMSO) for subsequent dilution in aqueousreaction mixtures with cell lysates and other biological samples. Theinventive compounds' reaction chemistry occurs effectively in simplebuffer conditions, they do not require accessory reagents such as copperor reducing agents, and they do not interfere with fluorescenceapplications. The inventive compounds are chemoselective in that thephosphine reactive group is specific in biological samples forazide-tagged molecules, ensuring that fluorescent labeling is specific.The inventive compounds exhibit high-performance fluorescence, e.g., 550Compound 1-phosphine and 650 Compound 1-phosphine are intense, highlystable fluorophores for fluorescent detection.

The following examples illustrate the utility of the invention but donot limit the claim scope.

EXAMPLE 1 Synthesis of 4-methyl-5-oxohexane sulfonic acid used tosynthesize Example 2 compound2,3-dimethyl-3-(3-sulfopropyl)-3H-indole-5-sulfonic acid di-potassiumsalt

Sodium hydride (2.1 g, 80 wt %=69 mmol) was slurried in 10 ml dry THF.The suspension was cooled to 0° C. and a solution ofethyl-2-methylacetoacetate (10 g, 69 mmol) in 10 ml dry THF was addeddropwise. The solution was stirred at room temperature for one h. Asolution of 1,3-propanesultone (8.42 g, 69 mmol) in 10 ml dry THF wasadded dropwise. Once the addition was complete, the solution was stirredfor two h at 40° C. The solution was evaporated to dryness. The residuewas dissolved in 100 ml water. The aqueous solution was extracted twicewith ethylacetate, then 100 ml concentrated HCl was added and thesolution was refluxed for two hours. The solvent was evaporated invacuum. The residue was purified by column chromatography (silica,methanol/dichloromethane) to give 4-methyl-5-oxohexane sulfonic acid.Yield 10 g; MS (ESI−): 193.2 [M]⁻

EXAMPLE 2 Synthesis of2,3-dimethyl-3-(3-sulfopropyl)-3H-indole-5-sulfonic acid di-potassiumsalt used to synthesize Example 3 compound1-(2-methoxy-ethyl)-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumand Example 4 compound1-[2-(2-methoxy-ethoxy)-ethyl]-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumand Example 5 compound1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumand Example 6 compound1-(5-carboxypentyl)-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indolium

Ten g (51 mmol) 4-hydrazino-benzene sulfonic acid and 9.85 g (51 mmol)4-methyl-5-oxohexane sulfonic acid were dissolved in 50 ml acetic acid.The solution was heated at 140° C. for four h. The solvent wasevaporated in vacuum. The oily residue was dissolved in 20 ml methanol,then 50 ml of a saturated solution of KOH in 2-propanol was added toyield a yellow precipitate. The solid was filtered off and dried invacuum. Yield 11 g, MS (ESI−): 172.5 [M]²⁻

EXAMPLE 3 Synthesis of1-(2-methoxy-ethyl)-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumused to synthesize 550, 650, 755 Compound 1

A mixture of 5 g (12.4 mmol)2,3-dimethyl-3-(3-sulfopropyl)-3H-indole-5-sulfonic acid dipotassiumsalt and 5.89 g (25.6 mmol) 2-methoxyethyl-p-toluene sulfonate washeated under argon for 24 h. The residue was purified by columnchromatography (reversed phase silica, methanol/water, TFA).

Yield 2.3 g, MS (ESI−): 404.1 [M-H]⁻

EXAMPLE 4 Synthesis of1-[2-(2-methoxy-ethoxy)-ethyl]-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumused to synthesize 550, 650, 755 Compound 2

A mixture of 5 g (12.4 mmol)2,3-dimethyl-3-(3-sulfopropyl)-3H-indole-5-sulfonic acid dipotassiumsalt and 7.1 g (25.6 mmol) [2-(2-methoxyethoxyl)ethoxy]-p-toluenesulfonate was heated under argon for 24 h. The residue was purified bycolumn chromatography (reversed phase silica, methanol/water, TFA).Yield 2.0 g. MS (ESI−): 448.2 [M-H]⁻

EXAMPLE 5 Synthesis of1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumused to synthesize 550, 650, 755 Compound 3

A mixture of 5 g (12.8 mmol)2,3-dimethyl-3-(3-sulfopropyl)-3H-indole-5-sulfonic acid dipotassiumsalt and 8.14 g (25.6 mmol)[2-[2-(2-methoxyethoxyl)ethoxy]ethoxy]p-toluene sulfonate was heatedunder argon for 24 h. The residue was purified by column chromatography(reversed phase silica, methanol/water, TFA). Yield 1.9 g, MS (ESI−):492.1 [M-H]⁻

EXAMPLE 6 Synthesis of1-(5-carboxypentyl)-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumused to synthesize Example 7 compound1-(5-carboxypentyl)-3-methyl-2-((E)-2-phenylamino-vinyl)-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumand Example 8 compound1-(5-carboxypentyl)-3-methyl-2-((1E,3E)-4-phenylamino-buta-1,3-dienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand Example 9 compound1-(5-carboxypentyl)-3-methyl-2-((1E,3E,5E)-6-phenylamino-hexa-1,3,5-trienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indolium

Both 5 g (15.7 mmol) 6-hydrazino-naphthalene-1,3-disulfonic acid and4.93 g (25 mmol) 4-methyl-5-oxohexane sulfonic acid were dissolved in 50ml acetic acid. The solution was heated at 140° C. for four hours. Thesolvent was evaporated in a vacuum. The oily residue was dissolved in 20ml methanol, then 50 ml of a saturated solution of KOH in 2-propanol wasadded to yield a yellow precipitate. The solid was filtered off anddried in vacuum. Yield 4.1 g, MS (ESI−): 158.2 [M]³⁻

EXAMPLE 7 Synthesis of1-(5-carboxypentyl)-3-methyl-2-((E)-2-phenylamino-vinyl)-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumusedto synthesize 550 Compounds

A combination of 0.92 g (2 mmol)1-(5-carboxypentyl)-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 0.43 g (2.2 mmol) N,N′-diphenylformamidine was dissolved in 20 mlmethanol and stirred for 4 h under reflux. The solvent was removed undervacuum. The residue was washed carefully with ethyl acetate. A darkyellow solid was obtained which was processed without furtherpurification.

MS (ESI−): 563.1 [M-H]⁻

EXAMPLE 8 Synthesis of1-(5-carboxypentyl)-3-methyl-2-((1E,3E)-4-phenylamino-buta-1,3-dienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumused to synthesize 650 Compounds

A combination of 0.92 g (2 mmol)1-(5-carboxypentyl)-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 0.57 g (2.2 mmol) malonaldehyde-bisphenylimine-hydrochloride wasdissolved in 10 ml acetic acid and 10 ml acetic anhydride and stirredfor four h at 120° C. The solvent was removed under vacuum. The residuewas washed carefully with ethyl acetate. A dark brown solid was obtainedwhich was processed without further purification. MS (ESI−): 589.2[M-H]⁻

EXAMPLE 9 Synthesis of1-(5-carboxypentyl)-3-methyl-2-((1E,3E,5E)-6-phenylamino-hexa-1,3,5-trienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumusedto synthesize 755 Compounds

A combination of 0.92 g (2 mmol)1-(5-carboxypentyl)-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 0.63 g (2.2 mmol) glutacondianil-hydrochloride was dissolved in 10ml acetic acid and 10 ml acetic anhydride and stirred for four h at 120°C. The solvent was removed under vacuum. The residue was washedcarefully with ethyl acetate. A dark solid was obtained which wasprocessed without further purification. MS (ESI−): 615.2 [M-H]⁻

EXAMPLE 10 Synthesis of 550 Compound 12-{(E)-3-[1-(5-carboxypentyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-1,3-dihydro-indol-(2E)-ylidene]-propenyl}-1-(2-methoxy-ethyl)-3-methyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumtri sodium salt

Five hundred sixty-four mg (1 mmol)1-(5-carboxypentyl)-3-methyl-2-((E)-2-phenylamino-vinyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 404 mg (1 mmol)1-(2-methoxy-ethyl)-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumwere dissolved in 20 ml of acetic acid/acetic anhydride (1/1), followedby 200 mg sodium acetate. The solution was stirred under reflux for 15min. After cooling to room temperature, 20 ml diethylether was added.The resulting precipitate (mixture of the diastereomers 550 Compound 1(isomer 1) and 550 Compound 1 (isomer 2)) was extracted by suction,washed with ether, and dried.

The residue was purified by column chromatography (RP-18,acetonitrile/water and concentrated HCl) to separate the diastereomersfrom each other. The diastereomer that first eluted from the column wastermed diastereomer 1 (550 Compound 1 (isomer 1)). The diastereomer thateluted second from the column was termed diastereomer 2 (550 Compound 1(isomer 2)). The diastereomers were separated, followed byneutralization and evaporation. Purification of the singlediastereomeric compound was completed on a RP-18 column,acetonitrile/water. The corresponding fractions were pooled and thesolvent was removed by distillation. The two products (diastereomers 550Compound 1 (isomer 1) and 550 Compound 1 (isomer 2)) were dried in highvacuum.

550 Compound 1 (isomer 1):

yield: 12%

UV-vis (PBS): λmax=557 nm, λem=572 nm

MS (ESI−) [M/z]: 291.2 [M]³⁻; 448.3 [M+Na]²⁻

550 Compound 1 (isomer 2):

yield: 23%

UV-vis (PBS): λmax=557 nm, λem=572 nm

MS (ESI−) [M/z]: 291.1 [M]³⁻; 448.2 [M+Na]²⁻

EXAMPLE 11 Synthesis of 550 Compound 22-{(E)-3-[1-(5-carboxypentyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-1,3-dihydro-indol-(2E)-ylidene]-propenyl}-1-[2-(2-methoxy-ethoxy)-ethyl]-3-methyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumtri sodium salt

Both 1 mmol1-(5-carboxypentyl)-3-methyl-2-((E)-2-phenylamino-vinyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 1 mmol1-[2-(2-methoxy-ethoxy)-ethyl]-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumwere dissolved in 20 ml acetic acid/acetic anhydride (1/1) followed bythe addition of 200 mg sodium acetate. The solution was stirred underreflux for 15 min. After cooling to room temperature, 20 ml diethyletherwas added. The resulting precipitate (mixture of the diastereomers 550-1compound 2 and 550-2 compound 2) was extracted by suction, washed withether and dried.

The residue was purified by column chromatography (RP-18,acetonitrile/water and concentrated HCl), thereby separating thediastereomers from each other, as described in Example 10.

EXAMPLE 12 Synthesis of 550 Compound 32-{(E)-3-[1-(5-carboxypentyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-1,3-dihydro-indol-(2E)-ylidene]-propenyl}-1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-3-methyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumtri sodium salt

One mmol1-(5-carboxypentyl)-3-methyl-2-((E)-2-phenylamino-vinyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 1 mmol1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumwere dissolved in 20 ml acetic acid/acetic anhydride (1/1) followed bythe addition of 200 mg sodium acetate. The solution was stirred underreflux for 15 min. After cooling to room temperature, 20 ml diethyletherwas added. The resulting precipitate (mixture of the diastereomers 550-1compound 2 and 550-2 compound 2) was extracted by suction, washed withether and dried.

The residue was purified by column chromatography (RP-18,acetonitrile/water and concentrated HCl), thereby separating thediastereomers from each other, as described in Example 10.

EXAMPLE 13 650 Compound 1 Synthesis of2-{(1E,3E)-5-[1-(5-carboxypentyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-1,3-dihydro-indol-(2E)-ylidene]-penta-1,3-dienyl}-1-(2-methoxy-ethyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumtri sodium salt

Both 90 mg (1 mmol)1-(5-carboxypentyl)-3-methyl-2-((1E,3E)-4-phenylamino-buta-1,3-dienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 404 mg (1 mmol)1-(2-methoxy-ethyl)-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumwere dissolved in 20 ml acetic acid/acetic anhydride (1/1) followed bythe addition of 200 mg of sodium acetate. The solution was stirred underreflux for 15 min. After cooling to room temperature, 20 ml diethyletherwas added. The resulting precipitate (mixture of the diastereomers 650Compound 1 (isomer 1) and 650 Compound 1 (isomer 2)) was extracted bysuction, washed with ether, and dried.

The residue was purified by column chromatography (RP-18,acetonitrile/water and concentrated HCl) to separate the diastereomersfrom each other. The diastereomer that first eluted from the column wastermed diastereomer 1 (650 Compound 1 (isomer 1)). The diastereomer thateluted second from the column was termed diastereomer 2 (650 Compound 1(isomer 2)). The diastereomers were separated, followed byneutralization and evaporation. Purification of the singlediastereomeric compound was completed on a RP-18 column,acetonitrile/water. The corresponding fractions were pooled and thesolvent was removed by distillation. The two products (diastereomers 650Compound 1 (isomer 1) and 650 Compound 1 (isomer 2)) were dried in highvacuum.

650 Compound 1 (isomer 1):

yield: 11%

UV-vis (PBS): λmax=654 nm, λem=672 nm

MS (ESI−) [M/z]: 299.7 [M]³⁻; 461.0 [M+Na]²⁻

650 Compound 1 (isomer 2):

yield: 24%

UV-vis (PBS): λmax=654 nm, λem=672 nm

MS (ESI−) [M/z]: 299.6 [M]³⁻; 461.1 [M+Na]²⁻

EXAMPLE 14 650 Compound 2 Synthesis of2-{(1E,3E)-5-[1-(5-carboxypentyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-1,3-dihydro-indol-(2E)-ylidene]-penta-1,3-dienyl}-1-[2-(2-methoxy-ethoxy)-ethyl]-3-methyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumtri sodium salt

Both 564 mg (1 mmol)1-(5-carboxypentyl)-3-methyl-2-((1E,3E)-4-phenylamino-buta-1,3-dienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 449 mg (1 mmol)1-[2-(2-methoxy-ethoxy)-ethyl]-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumwere dissolved in 20 ml of acetic acid/acetic anhydride (1/1) followedby the addition of 200 mg of sodium acetate. The synthesis and work-upwere carried out according to Example 13.

650-1 compound 2:

yield: 11%

UV-vis (PBS): λmax=654 nm, λem=672 nm

MS (ESI−) [M/z]: 314.4 [M]³⁻; 483.0 [M+Na]²⁻

650-2 compound 2:

yield: 16%

UV-vis (PBS): λmax=654 nm, λem=672 nm

MS (ESI−) [M/z]: 314.5 [M]³⁻; 483.1 [M+Na]²⁻

EXAMPLE 15 650 Compound 3 Synthesis of2-{(1E,3E)-5-[1-(5-carboxypentyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-1,3-dihydro-indol-(2E)-ylidene]-penta-1,3-dienyl}-1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-3-methyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumtri sodium salt—650 compound 3

Both 564 mg (1 mmol)1-(5-carboxypentyl)-3-methyl-2-((1E,3E)-4-phenylamino-buta-1,3-dienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 493 mg (1 mmol)1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumwere dissolved in 20 ml acetic acid/acetic anhydride (1/1) followed bythe addition of 200 mg sodium acetate. The synthesis and work-up werecarried out according to Example 13.

650-1 compound 3:

yield: 10%

UV-vis (PBS): λmax=654 nm, λem=672 nm

MS (ESI−) [M/z]: 329.2 [M]³⁻; 505.0 [M+Na]²⁻

650-2 compound 3:

yield: 23%

UV-vis (PBS): λmax=654 nm, λem=672 nm

MS (ESI−) [M/z]: 329.1 [M]³⁻; 505.1 [M+Na]²⁻

EXAMPLE 16 Synthesis of 755 Compound 12-{(1E,3E,5E)-7-[1-(5-Carboxypentyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-1,3-dihydro-indol-(2E)-ylidene]-hepta-1,3,5-trienyl}-1-(2-methoxy-ethyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumtri sodium salt

Six hundred and sixteen mg (1 mmol)1-(5-Carboxypentyl)-3-methyl-2-((1E,3E,5E)-6-phenylamino-hexa-1,3,5-trienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 404 mg (1 mmol)1-(2-methoxy-ethyl)-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumwere dissolved in 20 ml of acetic acid/acetic anhydride (1/1) followedby the addition of 200 mg of sodium acetate. The solution was stirredunder reflux for 15 min. After cooling to room temperature, 20 mldiethylether was added. The resulting precipitate (mixture of thediastereomers 755 Compound 1 (isomer 1) and 755 Compound 1 (isomer 2))was extracted by suction, washed with ether, and dried.

The residue was purified by column chromatography (RP-18,acetonitrile/water and concentrated HCl) to separate the diastereomersfrom each other. The diastereomer that first eluted from the column wastermed diastereomer 1 (755 Compound 1 (isomer 1)). The diastereomer thateluted second from the column was termed diastereomer 2 (755 Compound 1(isomer 2)). The diastereomers were separated, followed byneutralization and evaporation. Purification of the singlediastereomeric compound was completed on a RP-18 column,acetonitrile/water. The corresponding fractions were pooled and thesolvent was removed by distillation. The two products (diastereomers 755Compound 1 (isomer 1) and 755 Compound 1 (isomer 2)) were dried in highvacuum.

755 Compound 1 (isomer 1):

yield: 8%

UV-vis (PBS): λ_(max)=752 nm; λ_(em)=778 nm

MS (ESI−) [M/z]: 308.4 [M]³⁻; 474.2 [M+Na]²⁻

755 Compound 1 (isomer 2):

yield: 16%

UV-vis (PBS): λ_(max)=752 nm; λ_(em)=778 nm

MS (ESI−) [M/z]: 308.4 [M]³⁻; 474.2 [M+Na]²⁻.

EXAMPLE 17 Synthesis of 755 Compound 22-{(1E,3E,5E)-7-[1-(5-Carboxypentyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-1,3-dihydro-indol-(2E)-ylidene]-hepta-1,3,5-trienyl}-1-(2-methoxy-ethoxy)-3-methyl-5-sulfo-3-(3-sulfopropyl)-3H-indolium

Both 1 mmol1-(5-carboxypentyl)-3-methyl-2-((1E,3E,5E)-6-phenylamino-hexa-1,3,5-trienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 1 mmol1-[2-(2-methoxy-ethoxy)-ethyl]-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumwere dissolved in 20 ml acetic acid/acetic anhydride (1/1) followed bythe addition of 200 mg sodium acetate. The solution was stirred underreflux for 15 min. After cooling to room temperature, 20 ml diethyletherwas added. The resulting precipitate (mixture of the diastereomers 755compound 2 (isomer 1) and 755 compound 2 (isomer 2)) was extracted bysuction, washed with ether, and dried: The residue is purified by columnchromatography (RP-18, acetonitrile/water and concentrated HCl), therebyseparating the diastereomers from each other, as described in Example16.

EXAMPLE 18 Synthesis of 755 Compound 32-{(1E,3E,5E)-7-[1-(5-Carboxypentyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-1,3-dihydro-indol-(2E)-ylidene]-hepta-1,3,5-trienyl}-1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-3-methyl-5-sulfo-3-(3-sulfopropyl)-3H-indolium

Both 1 mmol1-(5-carboxypentyl)-3-methyl-2-((1E,3E,5E)-6-phenylamino-hexa-1,3,5-trienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 1 mmol1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumwere dissolved in 20 ml acetic acid/acetic anhydride (1/1) followed bythe addition of 200 mg sodium acetate. The solution was stirred underreflux for 15 min. After cooling to room temperature, 20 ml diethyletherwas added. The resulting precipitate (mixture of the diastereomers 755compound 3 (isomer 1) and 755 compound 3 (isomer 2)) was extracted bysuction, washed with ether and dried. The residue is purified by columnchromatography (RP-18, acetonitrile/water and concentrated HCl), therebyseparating the diastereomers from each other, as described in Example16.

EXAMPLE 19 Synthesis of 650 Compound 1-Phosphine

Sixty (60) mg (62 μmol) of Compound 1 were dissolved in a mixture of 3ml DMF and 300 μl water. After cooling to 0° C., 19 mg (62 μmol) TSTUand 11 μl (62 μmol) diisopropylethylamine were added. The reactionmixture was stirred for one hour at 0° C. After warming to roomtemperature, 82 mg (620 μmol) of ethylenediamine dihydrochloride and 53μl diisopropylethylamine (310 μmol) were added. The reaction mixture wasstirred for two hours. The solvent was then evaporated in vacuum. Theresidue was purified by HPLC chromatography. Yield: 50 mg of Compound1—aminomodified.

Fifty (50) mg (50.5 μmol) of Compound 1—aminomodified were dissolved in3 ml DMF, 23 mg (50.5 μmol) of phosphine-reagent-NHS-ester (Pierce) and26 μl (150 μmol) diisopropylethylamine were added. The reaction mixturewas stirred for two hours at room temperature. The solvent was removedunder vacuum and the residue was purified by HPLC chromatography. Yield:40 mg

EXAMPLE 20

Absorption profiles for inventive and commercial compounds weredetermined (FIG. 5) where DyLight 549-phosphine (blue), 550 Compound1-phosphine (purple), DyLight 649-phosphine (red), and 650 Compound1-phosphine (green) showed similar profiles in their respective pairs(baseline (100% transmission)). Maximum absorbance of 650 Compound1-phosphine was shifted 3 nm compared to DyLight 649-phosphine.

Fluorescence intensity was measured for DyLight 549-phosphine (blue) and550 Compound 1-phosphine (green) (FIG. 6) at an excitation/emission of560 nm/575 nm and 562 nm/576 nm, respectively; and for DyLight649-phosphine (blue) and 650 Compound 1-phosphine (green) (FIG. 7) at anexcitation/emission of 645 nm/672 nm and 646 nm/674 nm, respectively.The fluorescence intensities for the inventive compounds were similar totheir respective counterpart, and maximum fluorescence for 650 Compound1-phosphine was shifted 3 nm compared to DyLight 649-phosphine. Inaddition, the inventive phosphine-containing compounds exhibitedsolubility in dimethylsulfoxide (DMSO) at 10 mM.

EXAMPLE 21

Labeling efficiency of various inventive compounds was examined. Goatanti-Rabbit (GAR) antibodies were conjugated with NHS-PEG₄-azide at a10× molar ratio. Inventive and commercial compounds, each as thephosphine, were conjugated to azide-PEG₄-GAR goat anti-rabbit (GAR) forthree hours at 37° C. at a 10× molar ratio. Fluorescence Intensity andfunctional performance of the resulting compound-PEG₄-GAR conjugateswere determined.

Fluorescence intensity was measured for compound-PEG₄-GAR conjugateswhere the compound was DyLight 549-phosphine (blue), 550 Compound1-phosphine (green), DyLight 649-phosphine (yellow) and 650 Compound1-phosphine (red) (baseline (100% transmission)). FIG. 8 showingabsorbance scan of the conjugates after removal of the unconjugateddyes.

The conjugates were scanned and moles of dye per mole of protein (D/P)was calculated. Labeling efficiency was compared for compound-PEG₄-GARconjugates where the compound was DyLight 549-phosphine (left group;blue), 550 Compound 1-phosphine (left group; red), DyLight 649-phosphine(right group; blue) and 650 Compound 1-phosphine (right group; red)(FIG. 9) and for compound-PEG₄-GAM conjugates where the compound wasDyLight 549-phosphine (left group; blue), 550 Compound 1-phosphine (leftgroup; red), DyLight 649-phosphine (right group; blue) and 650 Compound1-phosphine (right group; red) (FIG. 10). The antibody was derivatizedwith NHS-PEG₄-azide. After removal of the unreacted NHS-azide, the 550Compound 1- or 650 Compound 1-phosphine was allowed to react with theazide labeled antibody. The reaction was carried out for 2-3 hours andthen the free dye was removed using the pierce Dye Removal Resin.Absorbance scans were performed and the mole dye to mole protein rationwas determined. 550 Compound 1-phosphine showed 25% increase and 650Compound 1-phosphine showed 10% increase over DyLight 549 or DyLight649, respectively, in the GAR-containing conjugates. 550 Compound1-phosphine showed a 2% decrease and 650 Compound 1-phosphine showed 10%increase over DyLight 549 or DyLight 649, respectively, in theGAM-containing conjugates. The results showed slightly higher dyeincorporation with both 550 Compound 1 and 650 Compound 1 compared toDyLight 549 and DyLight 649, respectively.

EXAMPLE 22

Performance of compound-GAM conjugates and compound-GAR conjugates wasevaluated in a functional assay. Wells of a 96 white opaque plate werecoated with target proteins mouse IgG immunoglobulin or rabbit IgGimmunoglobulin. One hundred μl mouse or rabbit IgG at a concentration of10 μg/ml was applied to the corresponding wells in columns 1 and 2. Thetarget proteins were serially diluted 1:1 from the wells in columns 2 to11 using 100 μl PBS. One hundred μl of samples from the wells in column11 were discarded. One hundred μl PBS was added to the wells in column12. The plates were incubated overnight at 4° C. and then blocked 2×200μl with Thermo Scientific SuperBlock® Blocking Buffer. The coated plateswere washed 2×200 μl with PBS-Tween and 1×200 μl with PBS. Conjugatesdiluted in PBS to 4 μg/ml were added to the corresponding plates (100μl/well) and then incubated for one h in the dark. The plates werewashed with 2×200 μl with PBS-Tween and 1×200 μl with PBS and filledwith PBS buffer (100 μl/well) prior to scanning on Tecan Safire using562 nm_(excitation)/576 nm_(emission) to detect fluorescence intensityand DyLight 649 and DyLight 650 plated were measured using 654nm_(excitation)/673 nm_(emission).

As shown in FIGS. 11-12, relative fluorescence units (RFU) were comparedfor compound-PEG₄-GAR conjugates where the compound was DyLight549-phosphine (red; FIG. 11), 550 Compound 1-phosphine (blue; FIG. 11),DyLight 649-phosphine (red; FIG. 12) and 650 Compound 1-phosphine (blue;FIG. 12) at various concentrations; the experiment was performed induplicate.

As shown in FIGS. 13-14, the fluorescence intensity, also termed theRFU, was compared for compound-PEG₄-GAM conjugates where the compoundwas DyLight 549-phosphine (blue; FIG. 13), 550 Compound 1-phosphine(red; FIG. 13), DyLight 649-phosphine (red; FIG. 14) and 650 Compound1-phosphine (blue; FIG. 14) at various concentrations. The results forDyLight 549 and 550 Compound 1 were similar. The results for DyLight 649and 650 Compound 1 were similar.

SDS-PAGE was performed (with minimal protein reduction) as shown inFIGS. 15 and 16 with 10 μg of non-reduced antibody loaded in each lane.The dye labeled samples were prepared in non-reducing LDS sample buffer,heated to 95° C. for 5 min. The samples were applied to a 4-12% Bis-Trisgel and subjected to electrophoresis. The gels were then scanned usingthe Epson Scanner (FIG. 15), then stained with Imperial Protein Stainfor one hour, destained, and then scanned (FIG. 16). The results showedthat azido-PEG4 conjugated with GAR reacted with dye-phosphines.

GAR conjugates in gel is shown in FIGS. 17 and 18, and fluorescenceshown in FIG. 19. Sample preparation and gel electrophoresis was thesame as described for FIGS. 15-16. The results show coupling of the dyeonly when the antibody is derivatized with an azido group. Gels werescanned using Typhoon 9410 imager with the Cy3 laser and Cy5 lasersettings.

EXAMPLE 23

The inventive compounds were evaluated for stability compared tocommercial dyes. All compounds were packed under argon as 1 mg portionsin plastic vials. The vials were sealed under argon with a drying pad inan aluminium coated pouch, and then stored at 50° C. for 14 days. Theresults of the stability study for selected compounds are shown belowwhere the determination of purity was by HPLC before and after storage.

550 Compound 1-Phosphine

Purity Unit size Before thermal stress After thermal stress 1 mg 96% 96%

650 Compound 1-Phosphine

Purity Unit size Before thermal stress After thermal stress 1 mg 97% 96%

The inventive compounds exhibited >90% stability by HPLC from variouslots.

Conjugate stability was also assessed. 550 Compound 1- and 650 Compound1-phosphines were conjugated to azido-PEG₄-GAM, subjected to 50° C. forfive days, and examined by Western blot. As shown in FIGS. 20 and 21,and a plot of the conjugate fluorescent intensity shown in FIG. 22,where each lane was loaded with 3 μg of antibody. The inventivecompound-phosphine derivatives are stable for >20 months at −20° C.

The phosphine-containing compounds stability, in the presence ofdimethylsulfoxide (DMSO) were also examined. Phosphine-containingcompounds were kept in DMSO solution at 37° C. for varying numbers ofdays, and then conjugated with azido-PEG4-GAM. The 550 Compound1-phosphine-PEG₄-GAM conjugates (FIG. 23) and 650 Compound1-phosphine-PEG₄-GAM conjugates (FIG. 24) were subjected to Western blotafter 0 day (lane 1), 1 day (lane 2), 2 days (lane 3), and 3 days (lane4). The resultant fluorescence intensity of the various conjugates fromFIGS. 23 and 24 are shown in FIG. 25. The relative fluorescence units(RFU) of conjugates formed from the phosphine-containing compounds inDMSO for 0 day (red square), 1 day (blue triangle), 3 days (yellowtriangle), and 6 days (green diamond), as a function of the amount ofantibody, are shown for 550 Compound 1-phosphine-PEG₄-GAM conjugates(FIG. 26) and for 650 Compound 1-phosphine-PEG₄-GAM conjugates (FIG.27). The EC₅₀ for each conjugate is also presented. The results indicatethat the phosphine-containing compounds are stable in DMSO for at leasta week.

EXAMPLE 24

The inventive and commercial compounds were examined in cell labelingexperiments using azido-sugars and adenocarcinomic human alveolar basalepithelial (A549) cells, human osteosarcoma (U2OS) cells, andimmortalized proximal tubule epithelial (HK2) cells. As is known in theart, introduction of labeled sugar moieties results in incorporation ofthe labeled sugar moieties in various cellular molecules and locations.

Metabolically incorporated azido-labeled sugars, N-azidoacetylgalactosamine, N-azidoacetyl glucosamine, and N-azidoacetyl mannosaminewas detected in vivo. Cells were incubated with 40 μM azido-sugar incell culture media for 72 hours and the live cells were incubated with100 μM of dye-labeled phosphine. The cells were then washed, fixed with4% paraformaldehyde and counterstained with Hoechst 33342 which labelsnuclei. For fixed cells, the cells were incubated with the azido-sugar,fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100,and incubated with 100 μM of dye-labeled phosphine. In live celllabeling using the phosphine labeling reaction used 100 μM of thedye-labeled phosphine for three hours at 37° C., and the CLICK-iT®labeling reaction, using DIBO-Alexa dye, used 30 μM of the DIBO Alexadye for one hour at room temperature (RT). In fixed cell labeling, thephosphine labeling reaction used 100 μM of the dye-labeled phosphine forthree hours at 37° C., and the CLICK-IT® labeling reaction, usingDIBO-Alexa dye, used 50 μM of the DIBO Alexa dye for one hour at RT inthe presence of 5 μM copper for one hour at RT in the absence of copper.

Either live (column A) or fixed (column B) A549 cells were labeled withN-azidoacetyl galactosamine, followed by labeling with 550 Compound1-phosphine (row 1), 650 Compound 1-phosphine (row 2), or CopperlessCLICK-iT® using DIBO-Alexa647 (row 3). Fixed A549 cells were labeledwith N-azidoacetyl galactosamine followed by labeling using CLICK-iT®reaction using copper with Alexa555-alkyne at either 5 μM for one hourat RT (row 4, column A) or 50 μM for one hour at RT (row 4, column B),as shown in FIG. 28.

Fluorescence intensity was measured in each spot identified using aspecific Bio-Application with the ArrayScanVTI image analysis software.A graph depicting spot average intensity for the live cell images ofFIG. 28, is shown for the 550 Compound 1-phosphine labeled cells (FIG.29A, wells 1-48), the 650 Compound 1-phosphine labeled cells (FIG. 29B,wells 1-48), and the DIBO-Alexa647 labeled cells (FIG. 29B, wells49-96). A graphical representation of spot average intensity for theimages of FIG. 28 is shown for live (FIG. 30, with two Y axes 550Compound 1 (red) spans up to 700; 650 Compound 1 spans up to 1600) andfixed (FIG. 31) cell labeling. The results showed that, in live cells,650 Compound 1-phosphine was better than Alexa647-DIBO CLICK-iT®labeling.

A549 cells, either live (column A) or fixed (column B), were labeledwith N-azidoacetyl glucosamine followed by labeling with 550 Compound1-phosphine (row 1), 650 Compound 1-phosphine (row 2), or CopperlessCLICK-IT® using DIBO-Alexa647 (row 3), as shown in FIG. 32. A graphdepicting spot average intensity for the live cell images of FIG. 32 isshown for the 550 Compound 1-phosphine labeled cells (FIG. 33A, wells1-48), the 650 Compound 1-phosphine labeled cells (FIG. 33B, wells1-48), and the DIBO-Alexa647 labeled cells (FIG. 33B, wells 49-96). Agraphical representation of the spot average intensity for the images ofFIG. 32 is shown for labeling live (FIG. 34) and fixed (FIG. 35) cells.

A549 cells, either live (column A) or fixed (column B), were labeledwith N-azidoacetyl mannosamine followed by labeling with 550 Compound1-phosphine (row 1), 650 Compound 1-phosphine (row 2), or CopperlessCLICK-iT® using DIBO-Alexa647 (row 3), as shown in FIG. 36. The patternof specific staining should typically be in the membrane, as seen inlive cells with dye-phosphine, unlike the deemed “non-specific”nuclear/whole cells in fixed cells when using the Click iT DIBO Alexa647. A graph depicting spot average intensity for the live cell imagesof FIG. 36, is shown for the 550 Compound 1-phosphine labeled cells(FIG. 37A, wells 1-48), the 650 Compound 1-phosphine labeled cells (FIG.37B, wells 1-48), and the DIBO-Alexa647 labeled cells (FIG. 37B, wells49-96). A graphical representation of the spot average intensity for theimages of FIG. 36 is shown for live (FIG. 38) and fixed (FIG. 39) cells.

U2OS cells, either live (column A) or fixed (column B), were labeledwith N-azidoacetyl galactosamine followed by labeling with 550 Compound1-phosphine (row 1), 650 Compound 1-phosphine (row 2), or CopperlessCLICK-iT® using DIBO-Alexa647 (row 3), as shown in FIG. 40. A graphdepicting spot average intensity for the live cell images of FIG. 40, isshown for the 550 Compound 1-phosphine labeled cells (FIG. 41A, wells1-48), the 650 Compound 1-phosphine labeled cells (FIG. 41B, wells1-48), and the DIBO-Alexa647 labeled cells (FIG. 41B, wells 49-96). Agraphical representation of the spot average intensity for the images ofFIG. 40 is shown for labeling live (FIG. 42) and fixed (FIG. 43) cells.As above, the results show that staining in live cells is specific inthe membrane, while fixed cells show non-specific staining in thenucleus.

U2OS cells, either live (column A) or fixed (column B), were labeledwith N-azidoacetyl glucosamine followed by labeling with 550 Compound1-phosphine (row 1), 650 Compound 1-phosphine (row 2), or CopperlessCLICK-iT® using DIBO-Alexa647 (row 3), as shown in FIG. 44. A graphdepicting spot average intensity for the live cell images of FIG. 44, isshown for the 550 Compound 1-phosphine labeled cells (FIG. 45A, wells1-48), the 650 Compound 1-phosphine labeled cells (FIG. 45B, wells1-48), and the DIBO-Alexa647 labeled cells (FIG. 45B, wells 49-96). Agraphical representation of the spot average intensity for the images ofFIG. 44 is shown for labeling live (FIG. 46) and fixed (FIG. 47) cells.As above, the results show that in live cells, 650 Compound 1-phosphinewas better than Alexa647-DIBO CLICK-iT® labeling.

U2OS cells, either live (column A) or fixed (column B), were labeledwith N-azidoacetyl mannosamine followed by labeling with 550 Compound1-phosphine (row 1), 650 Compound 1-phosphine (row 2), or CopperlessCLICK-iT® using DIBO-Alexa647 (row 3), as shown in FIG. 48. A graphdepicting spot average intensity for the live cell images of FIG. 48, isshown for the 550 Compound 1-phosphine labeled cells (FIG. 49A, wells1-48), the 650 Compound 1-phosphine labeled cells (FIG. 49B, wells1-48), and the DIBO-Alexa647 labeled cells (FIG. 49B, wells 49-96). Agraphical representation of the spot average intensity for the images ofFIG. 48 is shown for labeling live (FIG. 50) and fixed (FIG. 51) cells.As above, the results show that in live cells, 650 Compound 1-phosphinewas better than Alexa647-DIBO CLICK-iT® labeling. Cell loss can beindicative of cell death due to toxicity, potentially related to copperoxidation in Alexa 555.

HK2 cells, either live (column A) or fixed (column B), were labeled withN-azidoacetyl galactosamine followed by labeling with 550 Compound1-phosphine (row 1), 650 Compound 1-phosphine (row 2), or CopperlessCLICK-iT® using DIBO-Alexa647 (row 3), as shown in FIG. 52. A graphicalrepresentation of the spot average intensity for the images of FIG. 52is shown for labeling live (FIG. 53) and fixed (FIG. 54) cells. Asabove, the results show that in live cells, 650 Compound 1-phosphine wasbetter than Alexa647-DIBO CLICK-iT® labeling.

HK2 cells, either live (column A) or fixed (column B), were labeled withN-azidoacetyl glucosamine followed by labeling with 550 Compound1-phosphine (row 1), 650 Compound 1-phosphine (row 2), or CopperlessCLICK-iT® using DIBO-Alexa647 (row 3), as shown in FIG. 55. A graphicalrepresentation of the spot average intensity for the images of FIG. 55is shown for labeling live (FIG. 56) and fixed (FIG. 57) cells. Asabove, the results show that in live cells, 650 Compound 1-phosphine wasbetter than Alexa647-DI BO CLICK-iT® labeling.

HK2 cells, either live (column A) or fixed (column B), were labeled withN-azidoacetyl mannosamine followed by labeling with 550 Compound1-phosphine (row 1), 650 Compound 1-phosphine (row 2), or CopperlessCLICK-iT® using DIBO-Alexa647 (row 3), as shown in FIG. 58. A graphicalrepresentation of the spot average intensity for the images of FIG. 58is shown for live cell labeling (FIG. 59) and for fixed cell labeling(FIG. 60). As above, the results show that in live cells, 650 Compound1-phosphine was better than Alexa647-DIBO CLICK-iT® labeling.

EXAMPLE 25

The inventive and commercial compounds were examined in A549 cell lysatelabeling experiments using azido-sugars. Cell extracts were preparedfrom A549 incubated with azido-sugar N-azidoacetylgalactosamine(ManNAz), N-azidoacetylglucosamine (GlcNAz) or N-azidoacetylmannosamine(GalNAz). The cell extracts were incubated with either 550 Compound1-phosphine, 650 Compound 1-phosphine, CLICK-iT® Alexa555-Alkyne, orCLICK-iT® Alexa Fluor 647 DIBO Alkyne and analyzed by SDS-PAGE. Theresulting lysates were separated by electrophoresis. 10 μg protein wasloaded onto a PAGE gel (4-12% Bis-Tris gel). FIG. 61 shows proteinstaining viewed using the Typhoon 9410 imager showing the conjugation ofazido sugars from the cell extract labeled with either N-azidoacetylgalactosamine (Az-GalNAc; lanes 2 and 3), N-azidoacetyl glucosamine(Az-GlcNAc; lanes 4 and 5), or N-azidoacetyl mannosamine (Az-ManNAc;lanes 6 and 7) and conjugated with either CLICK-iT® Alexa555-Alkyne(lanes 2, 4, and 6) or 550 Compound 1-phosphine (lanes 3, 5, and 7),along with a molecular weight ladder (lane 1), as shown in FIG. 61.

The resulting lysates were separated by electrophoresis. Proteinstaining is viewed using the Typhoon 9410 imager after labeling with thedyes that had been labeled with either N-azidoacetyl galactosamine(Az-GalNAc; lanes 2 and 3), N-azidoacetyl glucosamine (Az-GlcNAc; lanes4 and 5), or N-azidoacetyl mannosamine (Az-ManNAc; lanes 6 and 7) andconjugated with either DIBO Alexa647 (lanes 2, 4, and 6) or 650 Compound1-phosphine (lanes 3, 5, and 7), along with a molecular weight ladder(lane 1), as shown in FIG. 62. The samples incubated with 650 Compound1-phosphine showed a different labeling pattern for each of the threeincorporated azido-sugars, demonstrating that different types ofglycosylation were detected specifically.

FIGS. 63-64 graphically represent band intensity of FIGS. 61-62. FIG. 63lanes 1-6 correspond to Gal-Alexa 555, Gal-550 Compound 1, Glc-Alexa555, Glc-550 Compound 1, Man-Alexa 555, Man-550 Compound 1,respectively. FIG. 64 lanes 1-6 correspond to Gal-Alexa 647, Gal-650Compound 1, Glc-Alexa 647, Glc-650 Compound 1, Man-Alexa 647, Man-650Compound 1, respectively. Relative fluorescence intensity for the gelimage of FIG. 61 comparing Alexa555 (copper) and 550 Compound1-phosphine (FIG. 63) and for the gel image of FIG. 62 comparingAlexa647 (copper-less) and 650 Compound 1-phosphine (FIG. 64). Resultsshows that 550 Compound 1-phosphine was better than CLICK-iT® Alexa555-alkyne.

EXAMPLE 26

The inventive and commercial compounds were used to label secretoryvesicles in various cell types using azido-sugars. Labeling all celltypes (HK2, A549, and U2OS cells) showed successful secretory vesiclestaining with N-azidoacetyl mannosamine (Az-ManNAc) and conjugation with650 Compound 1-phosphine resulted in labeling of secretory vesicles, asshown in FIGS. 65A and 66A (yellow arrows). Co-localization of Man-650Compound 1-phosphine, and this labeling was co-localized with PGP9.5, asecreted protein, as shown in FIGS. 65B and 66B (red arrows).

EXAMPLE 27

The inventive compounds were used in direct protein labeling onnitrocellulose (NC). As shown in FIG. 67, the inventivephosphine-containing compounds detect protein at nanogram level. It isalso likely that blocking would reduce the background to achieve bettersensitivity.

Overall, 550 Compound 1- and 650 Compound 1-phosphines performed equalto or better than DyLight 549- and DyLight 649-phosphines, respectively.In live cell labeling, the inventive phosphines outperformed theCopperless DIBO-Alexa Fluor CLICK-iT® reaction. In fixed cell labeling,the inventive phosphines showed similar performance to the CopperlessDIBO-Alexa Fluor CLICK-iT® reaction. The original alkyne CLICK-iT®reaction is less desirable for both live cell and fixed cell labelingreactions.

The embodiments shown and described in the specification are notlimiting in any way. Therefore, various changes, modifications, oralterations to those embodiments may be made without departing from thespirit of the invention in the scope of the following claims. Allreferences cited are expressly incorporated by reference herein in theirentirety.

What is claimed is:
 1. A compound of general formula III

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl group, heteroaliphatic with terminal SO₃, aPEG group P-L¹-Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a polyethylene glycol group, where thepolyethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive, a sulfonamide group -L¹-SO₂NH—P-L¹-Z, and a caboxamidegroup -L¹-CONH—P-L¹-Z; each of R⁷ and R⁸ is the same or different and isindependently selected from either H, SO₃, sulfoalkyl, heteroaliphatic,heteroaliphatic with terminal SO₃, a PEG group P-L¹-Z where P isselected from an ethylene glycol group, a diethylene glycol group, and apolyethylene glycol group, where the polyethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, a sulfonamidegroup -L¹-SO₂NH—P-L¹-Z, or a caboxamide group -L¹-CONH—P-L¹-Z; each ofR9 and R10 is the same or different and is independently selected fromeither aryl groups, substituted aryl groups, or cycloalkyl groups; R11is an electrophilic group selected from the group consisting of acarboxylic acid, an alkyl ester, an aryl ester, a substituted arylester, an aldehyde, and amide, an aryl amide, an alkyl halide, athioester, a sulfonyl ester, and alkyl ketone, an aryl ketone, asubstituted aryl ketone, a halosulfonyl, a nitrile, and a nitro; L is alinking group that forms a covalent bond between the dye and thephosphine; L¹ is selected from a divalent linear (—(CH₂)_(o)—, o=0 to15), branched, or cyclic alkane group that can be substituted by atleast one atom selected from oxygen, substituted nitrogen, and/orsulfur; Z is selected from H, CH₃, an alkyl group, or a heteroalkylgroup; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s)needed to compensate the negative charge brought by the cyanine; m is aninteger from 0 to 5 inclusive; o is an integer from 0 to 12 inclusive;and n is an integer from 1 to 3 inclusive; with the proviso that atleast one of R¹, R², R⁵, R⁶, R⁷, and R⁸ contains a PEG group.
 2. Acompound of general formula IV

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl group, heteroaliphatic with terminal SO₃, aPEG group P-L¹-Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a polyethylene glycol group, where thepolyethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive, a sulfonamide group -L¹-SO₂NH—P-L¹-Z, and a caboxamidegroup -L¹-CONH—P-L¹-Z; each of R⁷ and R⁸ is the same or different and isindependently selected from either H, SO₃, sulfoalkyl, heteroaliphatic,heteroaliphatic with terminal SO₃, a PEG group P-L¹-Z where P isselected from an ethylene glycol group, a diethylene glycol group, and apolyethylene glycol group, where the polyethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, a sulfonamidegroup -L¹-SO₂NH—P-L¹-Z, and a caboxamide group -L¹-CONH—P-L¹-Z; each ofR9 and R10 is the same or different and is independently selected fromeither aryl groups, substituted aryl groups, or cycloalkyl groups; R11is an electrophilic group selected from the group consisting of acarboxylic acid, an alkyl ester, an aryl ester, a substituted arylester, an aldehyde, and amide, an aryl amide, an alkyl halide, athioester, a sulfonyl ester, and alkyl ketone, an aryl ketone, asubstituted aryl ketone, a halosulfonyl, a nitrile, and a nitro; L is alinking group that forms a covalent bond between the dye and thephosphine; L¹ is selected from a divalent linear (—(CH₂)_(o)—, o=0 to15), branched, or cyclic alkane group that can be substituted by atleast one atom selected from oxygen, substituted nitrogen, and/orsulfur; Z is selected from H, CH₃, an alkyl group, or a heteroalkylgroup Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s)needed to compensate the negative charge brought by the cyanine; m is aninteger from 0 to 5 inclusive; p is an integer from 1 to 6 inclusive;and n is an integer from 1 to 3 inclusive; with the proviso that atleast one of R¹, R², R⁵, R⁶, R⁷, and R⁸ contains a PEG group.
 3. Acompound having the formula

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl group, heteroaliphatic with terminal SO₃, aPEG group P-L¹-Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a polyethylene glycol group, where thepolyethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive, a sulfonamide group -L¹-SO₂NH—P-L¹-Z, and a caboxamidegroup -L¹-CONH—P-L¹-Z; each of R is the same or different and isindependently selected from either H, SO₃, sulfoalkyl, heteroaliphatic,heteroaliphatic with terminal SO₃, a PEG group P-L¹-Z where P isselected from an ethylene glycol group, a diethylene glycol group, and apolyethylene glycol group, where the polyethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, a sulfonamidegroup -L¹-SO₂NH—P-L¹-Z, or a caboxamide group -L¹-CONH—P-L¹-Z; each ofR9 and R10 is the same or different and is independently selected fromeither aryl groups, substituted aryl groups, or cycloalkyl groups; R11is an electrophilic group selected from the group consisting of acarboxylic acid, an alkyl ester, an aryl ester, a substituted arylester, an aldehyde, and amide, an aryl amide, an alkyl halide, athioester, a sulfonyl ester, and alkyl ketone, an aryl ketone, asubstituted aryl ketone, a halosulfonyl, a nitrile, and a nitro; L is alinking group that forms a covalent bond between the dye and thephosphine; L¹ is selected from a divalent linear (—(CH₂)_(o)—, o=0 to15), branched, or cyclic alkane group that can be substituted by atleast one atom selected from oxygen, substituted nitrogen, and/orsulfur; Z is selected from H, CH₃, an alkyl group, or a heteroalkylgroup; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s)needed to compensate the negative charge brought by the cyanine; m is aninteger from 0 to 5 inclusive; o is an integer from 0 to 12 inclusive;and n is an integer from 1 to 3 inclusive.
 4. A compound having theformula

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl group, heteroaliphatic with terminal SO₃, aPEG group P-L¹-Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a polyethylene glycol group, where thepolyethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive, a sulfonamide group -L¹-SO₂NH—P-L¹-Z, and a caboxamidegroup -L¹-CONH—P-L¹-Z; each R is the same or different and isindependently selected from either H, SO₃, sulfoalkyl, heteroaliphatic,heteroaliphatic with terminal SO₃, a PEG group P-L¹-Z where P isselected from an ethylene glycol group, a diethylene glycol group, and apolyethylene glycol group, where the polyethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, a sulfonamidegroup -L¹-SO₂NH—P-L¹-Z, and a caboxamide group -L¹-CONH—P-L¹-Z; each ofR9 and R10 is the same or different and is independently selected fromeither aryl groups, substituted aryl groups, or cycloalkyl groups; R11is an electrophilic group selected from the group consisting of acarboxylic acid, an alkyl ester, an aryl ester, a substituted arylester, an aldehyde, and amide, an aryl amide, an alkyl halide, athioester, a sulfonyl ester, and alkyl ketone, an aryl ketone, asubstituted aryl ketone, a halosulfonyl, a nitrile, and a nitro; L is alinking group that forms a covalent bond between the dye and thephosphine; L¹ is selected from a divalent linear (—(CH₂)_(o)—, o=0 to15), branched, or cyclic alkane group that can be substituted by atleast one atom selected from oxygen, substituted nitrogen, and/orsulfur; Z is selected from H, CH₃, an alkyl group, or a heteroalkylgroup Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s)needed to compensate the negative charge brought by the cyanine; m is aninteger from 0 to 5 inclusive; p is an integer from 1 to 6 inclusive;and n is an integer from 1 to 3 inclusive.
 5. A compound having theformula

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl group, or heteroaliphatic with terminal SO₃;each of R is the same or different and is independently selected fromeither H, SO₃, sulfoalkyl, heteroaliphatic, or heteroaliphatic withterminal SO₃; each of R9 and R10 is the same or different and isindependently selected from either aryl groups, substituted aryl groups,or cycloalkyl groups; R11 is an electrophilic group selected from thegroup consisting of a carboxylic acid, an alkyl ester, an aryl ester, asubstituted aryl ester, an aldehyde, and amide, an aryl amide, an alkylhalide, a thioester, a sulfonyl ester, and alkyl ketone, an aryl ketone,a substituted aryl ketone, a halosulfonyl, a nitrile, and a nitro; L isa linking group that forms a covalent bond between the dye and thephosphine; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s)needed to compensate the negative charge brought by the cyanine; m is aninteger from 0 to 5 inclusive; o is an integer from 0 to 12 inclusive;and n is an integer from 1 to 3 inclusive.
 6. A compound having theformula

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl group, or heteroaliphatic with terminal SO₃;each of R is the same or different and is independently selected fromeither H, SO₃, sulfoalkyl, heteroaliphatic, or heteroaliphatic withterminal SO₃; each of R9 and R10 is the same or different and isindependently selected from either aryl groups, substituted aryl groups,or cycloalkyl groups; R11 is an electrophilic group selected from thegroup consisting of a carboxylic acid, an alkyl ester, an aryl ester, asubstituted aryl ester, an aldehyde, and amide, an aryl amide, an alkylhalide, a thioester, a sulfonyl ester, and alkyl ketone, an aryl ketone,a substituted aryl ketone, a halosulfonyl, a nitrile, and a nitro; L isa linking group that forms a covalent bond between the dye and thephosphine; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s)needed to compensate the negative charge brought by the cyanine; m is aninteger from 0 to 5 inclusive; p is an integer from 1 to 6 inclusive;and n is an integer from 1 to 3 inclusive.
 7. A compound of claim 1selected from the group consisting of


8. A compound of claim 2 selected from the group consisting of


9. A compound of claim 3 selected from the group consisting of


10. The compound of claim 4 selected from the group consisting of


11. The compound of claim 5 selected from the group consisting of


12. The compound of claim 6 selected from the group consisting of


13. A method using the compound of claim 1 in a chemoselective ligationreaction with an azido compound. 14-18. (canceled)
 19. A method oflabeling at least one biomolecule, the method comprising providing acomposition comprising at least one excipient and the compound of claim1 in an effective concentration to the biomolecule under conditionssufficient for specifically binding the compound to the biomolecule, inthe absence of non-specific binding, resulting in labeling of thebiomolecule. 20-24. (canceled)
 25. The method of claim 19 furthercomprising reacting a linking moiety with the at least one biomoleculeto result in an at least one biomolecule-linking moiety conjugate, andreacting the compound with the at least one biomolecule-linking moietyconjugate to result in an at least one biomolecule-linkingmoiety-compound conjugate. 26-30. (canceled)
 31. The method of claim 25where the linking moiety is a heterobifunctional linking moiety having afirst terminal group reactive with an amine group and a second terminalazide. 32-36. (canceled)
 37. The method of claim 31 where the linkingmoiety comprises a polyethylene glycol (PEG) group between the first andthe second functional termini. 38-42. (canceled)
 43. A method ofdetecting at least one biomolecule, the method comprising providing acomposition comprising at least one excipient and the compound of claim1 in an effective concentration to at least one biomolecule underconditions sufficient for binding the compound to the biomolecule, anddetecting the biomolecule-bound compound. 44-48. (canceled)
 49. A kitcomprising a compound of claim 1 and instructions for performing achemoselective ligation reaction using the compound with an azidocompound. 50-54. (canceled)